1,228 results on '"Apoenzymes"'
Search Results
2. GLUTAMIC-ASPARTIC TRANSAMINASE. REACTION OF HOLOENZYME WITH SUBSTRATES AND OF APOENZYME WITH VITAMIN B6 DERIVATIVES.
- Author
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EVANGELOPOULOS AE and SIZER IW
- Subjects
- Animals, Swine, Apoenzymes, Aspartate Aminotransferases, Carbon Isotopes, Chemical Phenomena, Chemistry, Hydrogen-Ion Concentration, Myocardium, Pyridoxal Phosphate, Pyridoxine, Research, Spectrophotometry, Vitamin B 6
- Published
- 1965
3. Structural insight into substrate and product binding in an archaeal mevalonate kinase.
- Author
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Miller, Bradley R. and Kung, Yan
- Subjects
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MEVALONATE kinase deficiency , *GENETIC disorders , *METHANOSARCINA mazei , *METHANOSARCINA , *APOENZYMES - Abstract
Mevalonate kinase (MK) is a key enzyme of the mevalonate pathway, which produces the biosynthetic precursors for steroids, including cholesterol, and isoprenoids, the largest class of natural products. Currently available crystal structures of MK from different organisms depict the enzyme in its unbound, substrate-bound, and inhibitor-bound forms; however, until now no structure has yet been determined of MK bound to its product, 5-phosphomevalonate. Here, we present crystal structures of mevalonate-bound and 5-phosphomevalonate-bound MK from Methanosarcina mazei (MmMK), a methanogenic archaeon. In contrast to the prior structure of a eukaryotic MK bound with mevalonate, we find a striking lack of direct interactions between this archaeal MK and its substrate. Further, these two MmMK structures join the prior structure of the apoenzyme to complete the first suite of structural snapshots that depict unbound, substrate-bound, and product-bound forms of the same MK. With this collection of structures, we now provide additional insight into the catalytic mechanism of this biologically essential enzyme. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
4. Crystal structure of an inulosucrase from Halalkalicoccus jeotgali B3T-a halophilic archaeal strain
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Komal Ghauri, Nayla Munawar, Hazrat Ali, Russell Wallis, Munir A. Anwar, Tjaard Pijning, Muhammad Afzal Ghauri, and X-ray Crystallography
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0301 basic medicine ,Protein Folding ,Sucrose ,Protein Conformation ,Crystallography, X-Ray ,Biochemistry ,03 medical and health sciences ,Apoenzymes ,0302 clinical medicine ,Fructan ,Molecular Biology ,Gene ,chemistry.chemical_classification ,Halobacteriaceae ,Inulosucrase ,biology ,Strain (chemistry) ,Chemistry ,Cell Biology ,biology.organism_classification ,Archaea ,Halophile ,030104 developmental biology ,Enzyme ,Hexosyltransferases ,030220 oncology & carcinogenesis ,Trisaccharides ,Bacteria - Abstract
Several archaea harbour genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure-function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition and pharmaceutical applications. In this study, 3D structure of an inulin-type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form as well as with bound substrate (sucrose) molecule and first transglycosylation product (1-kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five-bladed β-propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram-negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram-positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram-negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1-kestose indicates that the residues D287 in the 4B-4C loop, Y330 in 4D-5A and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure-function studies of FTF enzymes, particularly those from archaea.
- Published
- 2021
5. Comparative protein structure network analysis on <scp> 3CL pro </scp> from <scp>SARS‐CoV</scp> ‐1 and <scp>SARS‐CoV</scp> ‐2
- Author
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Mohd Akif and Surabhi Lata
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Models, Molecular ,SARS coronavirus ,viruses ,medicine.medical_treatment ,Computational biology ,main protease (Mpro, 3CLpro) ,Biochemistry ,Structural variation ,03 medical and health sciences ,Apoenzymes ,Protein structure ,Structural Biology ,medicine ,Protease Inhibitors ,Binding site ,Protein Structure, Quaternary ,skin and connective tissue diseases ,Molecular Biology ,Research Articles ,Coronavirus 3C Proteases ,protein structure graph ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,Protease ,biology ,SARS-CoV-2 ,Chemistry ,fungi ,030302 biochemistry & molecular biology ,Active site ,Covid19 ,respiratory tract diseases ,Amino acid ,body regions ,Severe acute respiratory syndrome-related coronavirus ,biology.protein ,Protein quaternary structure ,protein structure network ,Protein Multimerization ,Holoenzymes ,Research Article ,Network analysis - Abstract
The main protease Mpro, 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS‐CoV‐1 and SARS‐CoV‐2; the inhibitors used to block the activity of SARS‐CoV‐1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS‐CoV‐2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3‐D crystallographic structure of Mpro from SARS‐CoV‐1 and SARS‐CoV‐2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS‐CoV‐1 and SARS‐CoV‐2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS‐CoV‐1 and SARS‐CoV‐2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros. Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.
- Published
- 2021
6. Structural insights into a new substrate binding mode of a histidine acid phosphatase from Legionella pneumophila
- Author
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Dan Zhou, Jing Li, Xiaoyu Qi, Honghua Ge, Hui Zhang, Qi Chen, Yan-Bin Teng, Yu Guo, Nannan Zhang, and Xiaofang Chen
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Models, Molecular ,0301 basic medicine ,Stereochemistry ,Phenylalanine ,Ribose ,Acid Phosphatase ,Biophysics ,Tartrate ,Biochemistry ,Legionella pneumophila ,Substrate Specificity ,Hydroxylation ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,0302 clinical medicine ,stomatognathic system ,Catalytic Domain ,Moiety ,Histidine ,Amino Acid Sequence ,Tartrates ,Molecular Biology ,biology ,Adenine ,Substrate (chemistry) ,Active site ,Cell Biology ,biology.organism_classification ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Mutation ,biology.protein ,Sequence Alignment ,Protein Binding - Abstract
MapA is a histidine acid phosphatase (HAP) from Legionella pneumophila that catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine. Several structures of HAPs, including MapA, in complex with the inhibitor tartrate have been solved and the substrate binding tunnel identified; however, the substrate recognition mechanism remains unknown. To gain insight into the mechanism of substrate recognition, the crystal structures of apo-MapA and the MapAD281A mutant in complex with 5′-AMP were solved at 2.2 and 2.6 A resolution, respectively. The structure of the MapAD281A/5′-AMP complex reveals that the 5′-AMP fits fully into the substrate binding tunnel, with the 2′-hydroxyl group of the ribose moiety stabilized by Glu201 and the adenine moiety sandwiched between His205 and Phe237. This is the second structure of a HAP/AMP complex solved with 5′-AMP binding in a unique manner in the active site. The structure presents a new substrate recognition mechanism of HAPs.
- Published
- 2021
7. Crystallographic and molecular dynamics simulation analysis of NAD synthetase from methicillin resistant Staphylococcus aureus (MRSA)
- Author
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Kazi Nasrin Sultana, Mohammad Imran Siddiqi, Sandeep Kumar Srivastava, and Jitendra Kuldeep
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Methicillin-Resistant Staphylococcus aureus ,Protein Conformation ,Stereochemistry ,In silico ,02 engineering and technology ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular dynamics ,Apoenzymes ,Amide Synthases ,Structural Biology ,Catalytic Domain ,Amide ,Enzyme Stability ,medicine ,Humans ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,Principal Component Analysis ,0303 health sciences ,biology ,Substrate (chemistry) ,Active site ,Hydrogen Bonding ,General Medicine ,NAD ,021001 nanoscience & nanotechnology ,Protein Subunits ,Enzyme ,chemistry ,Staphylococcus aureus ,biology.protein ,NAD+ kinase ,0210 nano-technology - Abstract
NAD synthetase (NadE) catalyzes the last step in NAD biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction with co-substrates ATP and amide donor ammonia. In this study, we report the crystal structure of Staphylococcus aureus NAD synthetase enzyme (saNadE) at 2.3 A resolution. We used this structure to perform molecular dynamics simulations of apo-enzyme, enzyme-substrate (NadE with ATP and NaAD) and enzyme-intermediate complexes (NadE with NaAD-AMP) to investigate key binding interactions and explore the conformational transitions and flexibility of the binding pocket. Our results show large shift of N-terminal region in substrate bound form which is important for ATP binding. Substrates drive the correlated movement of loop regions surrounding it as well as some regions distal to the active site and stabilize them at complex state. Principal component analysis of atomic projections distinguish feasible trajectories to delineate distinct motions in enzyme-substrate to enzyme-intermediate states. Our results suggest mixed binding involving dominant induced fit and conformational selection. MD simulation extracted ensembles of NadE could potentially be utilized for in silico screening and structure based design of more effective Methicillin Resistant Staphylococcus aureus (MRSA) inhibitors.
- Published
- 2020
8. Structure of serotonin receptors: molecular underpinning of receptor activation and modulation
- Author
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Alexander Dityatev, Evgeni Ponimaskin, and Markus Zweckstetter
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Cancer Research ,Underpinning ,Serotonin ,Protein Conformation ,QH301-705.5 ,chemistry [Apoenzymes] ,genetics [Apoenzymes] ,Molecular neuroscience ,Crystallography, X-Ray ,Ligands ,Apoenzymes ,Genetics ,chemistry [Serotonin] ,Humans ,ultrastructure [Apoenzymes] ,ddc:610 ,genetics [Serotonin] ,Biology (General) ,chemistry [Receptors, Serotonin] ,5-HT receptor ,genetics [Receptors, Serotonin] ,Chemistry ,Research Highlight ,chemistry [Serotonin Receptor Agonists] ,Serotonin Receptor Agonists ,Modulation ,Receptors, Serotonin ,ultrastructure [Receptors, Serotonin] ,Structural biology ,Medicine ,Receptor activation ,Neuroscience - Published
- 2021
9. Williams–Beuren syndrome‐related methyltransferase WBSCR27: cofactor binding and cleavage
- Author
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Olga I. Kechko, Vladimir I. Polshakov, Vladimir A. Mitkevich, Olga A. Petrova, Petr V. Sergiev, Sofia S. Mariasina, S.V. Efimov, Vladimir V. Klochkov, Chi-Fon Chang, and Olga A. Dontsova
- Subjects
0301 basic medicine ,S-Adenosylmethionine ,Methyltransferase ,Protein Conformation ,Biochemistry ,Cofactor ,Mice ,03 medical and health sciences ,Apoenzymes ,0302 clinical medicine ,Animals ,Molecular Biology ,Protein secondary structure ,chemistry.chemical_classification ,Cofactor binding ,Thionucleosides ,Deoxyadenosines ,biology ,Isothermal titration calorimetry ,Methyltransferases ,Cell Biology ,Methylation ,S-Adenosylhomocysteine ,Nucleosidases ,030104 developmental biology ,Enzyme ,chemistry ,030220 oncology & carcinogenesis ,biology.protein - Abstract
Williams-Beuren syndrome, characterized by numerous physiological and mental problems, is caused by the heterozygous deletion of chromosome region 7q11.23, which results in the disappearance of 26 protein-coding genes. Protein WBSCR27 is a product of one of these genes whose biological function has not yet been established and for which structural information has been absent until now. Using NMR, we investigated the structural and functional properties of murine WBSCR27. For protein in the apo form and in a complex with S-(5'-adenosyl)-l-homocysteine (SAH), a complete NMR resonance assignment has been obtained and the secondary structure has been determined. This information allows us to attribute WBSCR27 to Class I methyltransferases. The interaction of WBSCR27 with the cofactor S-(5'-adenosyl)-l-methionine (SAM) and its metabolic products - SAH, 5'-deoxy-5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'dAdo) - was studied by NMR and isothermal titration calorimetry. SAH binds WBSCR27 much tighter than SAM, leaving open the question of cofactor turnover in the methylation reaction. One possible answer to this question is the presence of weak but detectable nucleosidase activity for WBSCR27. We found that the enzyme catalyses the cleavage of the adenine moiety from SAH, MTA and 5'dAdo, similar to the action of bacterial SAH/MTA nucleosidases. We also found that the binding of SAM or SAH causes a significant change in the structure of WBSCR27 and in the conformational mobility of the protein fragments, which can be attributed to the substrate recognition site. This indicates that the binding of the cofactor modulates the folding of the substrate-recognizing region of the enzyme.
- Published
- 2020
10. Low-resolution SAXS and structural dynamics analysis on M. tuberculosis GmhB enzyme involved in GDP-heptose biosynthetic pathway
- Author
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Ashish, Ajay K. Saxena, Sumita Karan, Bhanu Pratap, and Shiv Pratap Singh Yadav
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Circular dichroism ,Heptose ,02 engineering and technology ,Guanosine Diphosphate ,Biochemistry ,03 medical and health sciences ,Apoenzymes ,X-Ray Diffraction ,Structural Biology ,Catalytic Domain ,Scattering, Small Angle ,Magnesium ,Amino Acid Sequence ,Binding site ,Molecular Biology ,030304 developmental biology ,Dehalogenase ,chemistry.chemical_classification ,0303 health sciences ,biology ,Small-angle X-ray scattering ,Active site ,Substrate (chemistry) ,Mycobacterium tuberculosis ,General Medicine ,Heptoses ,021001 nanoscience & nanotechnology ,Phosphoric Monoester Hydrolases ,Random coil ,Molecular Docking Simulation ,Zinc ,Crystallography ,chemistry ,biology.protein ,0210 nano-technology - Abstract
The M. tuberculosis GmhB protein converts the d-glycero-α-d-manno-heptose 1,7-bisphosphate (GMB) intermediate into d-glycero-α-d-manno-heptose 1-phosphate by removing the phosphate group at the C-7 position. To understand the structure and substrate binding mechanism, the MtbGmhB was purified which elutes as monomer on gel filtration column. The small angle x-ray scattering analysis shows that MtbGmhB forms fully folded monomer with shape profile similar to its modeled structure. The circular dichroism analysis shows 38% α-helix, 15% β-sheets and 47% random coil structures in MtbGmhB, similar to haloalkanoic acid dehalogenase (HAD) phosphohydrolase enzymes. The modeled MtbGmhB structure shows the catalytic site, which forms a concave, semicircular surface using the three loops around GMB substrate binding site. Dynamic simulation analysis on (i) Apo (ii) GMB bound (iii) GMB + Mg2+ bound (iv) Zn2+ +GMB + Mg2+ bound MtbGmhB structures show that Zn2+ as well as Mg2+ ions stabilize the loop conformation and trigger the changes in GMB substrate binding to active site of MtbGmhB. Upon demetallization, the large conformational changes occurred in ions binding loops, and leads to difference in GMB substrate binding to MtbGmhB. Our study provides information about structure and substrate binding of MtbGmhB, which may contribute in therapeutic development against M. tuberculosis.
- Published
- 2019
11. NMR solution structures of Runella slithyformis RNA 2'-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity
- Author
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Sébastien Alphonse, Stewart Shuman, Ankan Banerjee, Ranajeet Ghose, and Swathi Dantuluri
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Models, Molecular ,Stereochemistry ,Protein Conformation ,AcademicSubjects/SCI00010 ,Cytophagaceae ,NAR Breakthrough Article ,Biology ,010402 general chemistry ,Ligands ,01 natural sciences ,Phosphotransferase ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Bacterial Proteins ,Ribose ,Genetics ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Nicotinamide ,Adenosine diphosphate ribose ,Nucleotides ,Phosphotransferases ,RNA ,NAD ,0104 chemical sciences ,NAD binding ,chemistry ,Mutagenesis ,Nicotinamide riboside ,NAD+ kinase ,Protein Binding - Abstract
Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO4 to NAD+ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2′-PO4 (mimicking the substrate RNA 2′-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.
- Published
- 2021
12. Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase
- Author
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Opher Gileadi, T.J. Gorrie-Stone, Setayesh Yadzani, Yuliana Yosaatmadja, Louise Dunnett, Matthieu Schapira, José Brandão-Neto, Antony Aimon, Frank von Delft, Alice Douangamath, J.A. Newman, R. Skyner, and D. Fearon
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Models, Molecular ,Protein Conformation ,Science ,Druggability ,General Physics and Astronomy ,Viral Nonstructural Proteins ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Article ,Phosphates ,Structure-Activity Relationship ,03 medical and health sciences ,Protein structure ,Adenosine Triphosphate ,Apoenzymes ,0302 clinical medicine ,Nucleotide ,Amino Acid Sequence ,Enzyme Inhibitors ,Binding site ,Peptide sequence ,X-ray crystallography ,030304 developmental biology ,Sequence (medicine) ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,Binding Sites ,Molecular medicine ,biology ,SARS-CoV-2 ,Helicase ,General Chemistry ,Methyltransferases ,3. Good health ,Crystallography ,Viral replication ,chemistry ,030220 oncology & carcinogenesis ,Drug Design ,Proteome ,biology.protein ,Nucleic acid ,RNA, Viral ,Structural biology ,RNA Helicases ,030217 neurology & neurosurgery - Abstract
There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents., The SARS-CoV-2 NSP13 helicase is essential for viral replication and of interest as a drug target. Here, the authors present the crystal structures of NSP13 in the apo form and bound to either phosphate or the non-hydrolysable ATP analog AMP-PNP and discuss the helicase mechanism. They also perform a crystallographic fragment screening and identify 65 bound fragments, which could help in the design of new antiviral agents.
- Published
- 2021
13. Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution
- Author
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Ramya Rangan, Shanshan Li, Rhiju Das, Zhaoming Su, Kalli Kappel, Grigore D. Pintilie, Yuquan Wei, Bingnan Luo, Wah Chiu, Kaiming Zhang, and Michael Z. Palo
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Models, Molecular ,Conformational change ,Multidisciplinary ,biology ,Cryo-electron microscopy ,Chemistry ,Cryoelectron Microscopy ,Tetrahymena ,Ribozyme ,Intron ,RNA ,biology.organism_classification ,Article ,Tetrahymena thermophila ,Protein structure ,Apoenzymes ,Guanosine binding ,Biophysics ,biology.protein ,Nucleic Acid Conformation ,RNA, Catalytic ,Holoenzymes - Abstract
Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1–3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure–function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 A. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure–function relationships in ribozymes. Cryo-electron microscopy has been used to determine the structure of the Tetrahymena ribozyme (a catalytic RNA) at sufficiently high resolution to model side chains and metal ions.
- Published
- 2021
14. Time-dependent conformational analysis of ALK5-lumican complex in presence of graphene and graphene oxide employing molecular dynamics and MMPBSA calculation
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Santhosh Kumar Nagarajan, Sindhiya Sridharan, G. Devanand Venkatasubbu, and Kathirvel Venugopal
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Lumican ,0303 health sciences ,Graphene ,Chemistry ,030303 biophysics ,General Medicine ,Molecular Dynamics Simulation ,law.invention ,Molecular Docking Simulation ,Extracellular matrix ,03 medical and health sciences ,Molecular dynamics ,Apoenzymes ,Chondroitin Sulfate Proteoglycans ,Keratan Sulfate ,Structural Biology ,law ,Cell surface receptor ,Biophysics ,Graphite ,Receptor ,Wound healing ,Molecular Biology ,Binding selectivity - Abstract
Lumican, an extracellular matrix protein avails wound healing by binding to ALK5 membrane receptor (TGF-beta receptor I). Their interaction enables epithelialization and substantiates rejuvenation of injured tissue. To enrich permanence of ALK5-lumican interaction, we employed graphene and graphene oxide co-factors. Herein, this study explicates concomitancy of graphene and graphene oxide with ALK5-lumican. We performed an in silico approach involving molecular modelling, molecular docking, molecular dynamics for 200 ns, DSSP analysis and MMPBSA calculations. Results of molecular dynamics indicate cofactors influential in altering bioactive site of lumican than ALK5. Similarly, MMPBSA calculations unveiled binding energy of apoenzyme as −108.09 kcal/mol, holoenzyme (G) as −79.20 kcal/mol and holoenzyme (GO) as −114.33 kcal/mol. This concludes graphene oxide lucrative in enhancing binding energy of ALK5-lumican in holoenzyme (GO) via coil formation of Lum C13 domain. In contrast, graphene reduced binding energy of ALK5-lumican in holoenzyme (G) modifying Lum C13 into beta sheets. MMPBSA residual contribution analysis of Lum C13 residues revealed binding energy of −13.9 kcal/mol for apoenzyme, −6.8 kcal/mol for holoenzyme (G) and −19.5 kcal/mol for holoenzyme (GO). This supports coil formation propitious for better ALK5-Lum interaction. Highest SASA energy of −21.05 kcal/mol of holoenzyme (G) assures graphene reasonable for improved ALK5-lumican hydrophobicity. As per the motive of the study, graphene oxide enriches permanence of ALK5-lumican. This provides counsel for plausible exploitation of lumican and graphene oxide as targeted/nano drug delivery system to reinstate acute wounds, chronic wounds, corneal wounds, hypertrophic scars and keloids in near future. Communicated by Ramaswamy H. Sarma
- Published
- 2021
- Full Text
- View/download PDF
15. Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4
- Author
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Narayanan Manoj and Samar Ballabha Mohapatra
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Models, Molecular ,Glycoside Hydrolases ,Stereochemistry ,Protein Conformation ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Protein Structure, Secondary ,Substrate Specificity ,03 medical and health sciences ,Structure-Activity Relationship ,Apoenzymes ,Bacterial Proteins ,Glucuronic Acid ,Catalytic Domain ,Glycoside hydrolase ,Thermotoga maritima ,Binding site ,Molecular Biology ,Ternary complex ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Manganese ,biology ,030302 biochemistry & molecular biology ,Active site ,Glycosidic bond ,Cell Biology ,biology.organism_classification ,NAD ,Hyperthermophile ,Recombinant Proteins ,Dithiothreitol ,Kinetics ,Enzyme ,chemistry ,Multigene Family ,biology.protein ,Mutagenesis, Site-Directed ,Holoenzymes ,Protein Binding - Abstract
Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure–function relationships within the unique GH4 family.
- Published
- 2020
16. Antioxidant and antibacterial activities, interfacial and emulsifying properties of the apo and holo forms of purified camel and bovine α-lactalbumin
- Author
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Hamadi Attia, M.A. Ayadi, Roua Lajnaf, Mourad Jridi, Houda Gharsallah, École Nationale d'Ingénieurs de Sfax | National School of Engineers of Sfax (ENIS), Ingénierie des Agro-polymères et Technologies Émergentes (UMR IATE), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Ministry of Scientific Research and Technology of Tunisia
- Subjects
Antioxidant ,Camelus ,medicine.medical_treatment ,02 engineering and technology ,Biochemistry ,Antioxidants ,Ferrous ,03 medical and health sciences ,Apoenzymes ,Antioxidant activity ,Structural Biology ,medicine ,[CHIM]Chemical Sciences ,Animals ,Penicillium bilaiae ,Food science ,Molecular Biology ,Purification ,030304 developmental biology ,Lactalbumin ,0303 health sciences ,biology ,Chemistry ,Penicillium ,General Medicine ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Aspergillus sclerotiorum ,Anti-Bacterial Agents ,Emulsification ,Aspergillus ,13. Climate action ,Aspergillus tamari ,Camel alpha-lactalbumin ,Cattle ,Emulsions ,Antibacterial activity ,0210 nano-technology ,Holoenzymes ,Hydrophobic and Hydrophilic Interactions ,Bacteria - Abstract
International audience; The antioxidant and antibacterial activities of camel and bovine α-lactalbumin (α-La) in both calcium-loaded (holo) and calcium-depleted (apo) forms were investigated and compared. Antioxidant assay showed that camel and bovine α-La exhibited significant Ferric-reducing antioxidant power (FRAP), ferrous iron-chelating activity (FCA) and antiradical activities especially in their apo form. Camel apo α-La also exhibited attractive antibacterial activities against Gram-negative bacteria (Pseudomonas aeruginosa) and against fungal pathogens species (Penicillium bilaiae, Aspergillus tamari and Aspergillus sclerotiorum). Likewise, emulsifying properties (emulsification ability (EAI) and stability (ESI) indexes) and the surface characteristics (surface hydrophobicity, ζ-potential and interfacial tension) of the α-La were assessed. Maximum EAI were found at pH 7.0, with higher EAI values for the camel apo α-La (EAI ~19.5 m2/g). This behavior was explained by its relative high surface hydrophobicity and its greater efficiency to reduce the surface tension at the oil-water interface. Furthermore, emulsions were found to be more stable at pH 7.0 compared to pH 5.0 (ESI ~50%) due to the higher electrostatic repulsive forces between oil droplets at pH 7.0 in consistence with the ζ-potential results. This study concluded that the camel apo α-La has antibacterial, antioxidant, and emulsifying properties in agricultural and food industries.
- Published
- 2020
17. Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase
- Author
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Brian Broom-Peltz, Javier M. González, Ricardo Marti-Arbona, Julian C.-H. Chen, and Clifford J. Unkefer
- Subjects
Models, Molecular ,Oxaloacetic Acid ,Protein Conformation, alpha-Helical ,0301 basic medicine ,Conformational change ,Malates ,BIOFUELS ,Gene Expression ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,Research Communications ,purl.org/becyt/ford/1 [https] ,chemistry.chemical_compound ,Apoenzymes ,Malate Dehydrogenase ,Structural Biology ,Catalytic Domain ,Oxaloacetic acid ,Cloning, Molecular ,Ternary complex ,chemistry.chemical_classification ,biology ,Bioquímica y Biología Molecular ,Condensed Matter Physics ,Recombinant Proteins ,METHYLOBACTERIUM EXTORQUENS ,Methylobacterium extorquens ,Protons ,CIENCIAS NATURALES Y EXACTAS ,Protein Binding ,Catalytic complex ,Stereochemistry ,MALATE DEHYDROGENASE ,Genetic Vectors ,Biophysics ,Malate dehydrogenase ,Ciencias Biológicas ,03 medical and health sciences ,Bacterial Proteins ,Oxidoreductase ,Escherichia coli ,Genetics ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,purl.org/becyt/ford/1.6 [https] ,Adenosine Diphosphate Ribose ,030102 biochemistry & molecular biology ,Active site ,Hydrogen Bonding ,NAD ,biology.organism_classification ,Kinetics ,030104 developmental biology ,chemistry ,biology.protein ,METHYLOTROPHS ,Protein Multimerization - Abstract
Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.Crystal structures of apo malate dehydrogenase (MDH) from Methylobacterium extorquens, MDH bound to NAD+, and MDH with oxaloacetate and ADP-ribose revealed conformational changes, closing the active site upon coenzyme and substrate binding. In the ternary complex, His284 is in position to donate a proton in the formation of (2S)-malate. Fil: Gonzalez, Javier Marcelo. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; Argentina Fil: Marti Arbona, R.. Bioscience Division, Los Alamos National Laboratory; Estados Unidos Fil: Chen, J. C. H.. Bioscience Division, Los Alamos National Laboratory; Estados Unidos Fil: Broom Peltz, B.. Bioscience Division, Los Alamos National Laboratory; Estados Unidos Fil: Unkefer, C. J.. Bioscience Division, Los Alamos National Laboratory; Estados Unidos
- Published
- 2018
18. Activation Mechanism of the Streptomyces Tyrosinase Assisted by the Caddie Protein
- Author
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Naohiko Bando, Yasuyuki Matoba, Shogo Kihara, Shun Hirota, Miyuki Sakaguchi, Masanori Sugiyama, Teruo Kuroda, Hulin Tai, Takashi Ogura, Yoshimi Muraki, Hironari Yoshitsu, and Kure'e Kayama
- Subjects
Models, Molecular ,0301 basic medicine ,Reducing agent ,Stereochemistry ,Tyrosinase ,01 natural sciences ,Biochemistry ,Streptomyces ,Active center ,Protein Aggregates ,03 medical and health sciences ,Residue (chemistry) ,Apoenzymes ,Bacterial Proteins ,Catalytic Domain ,Benzoquinones ,Binding site ,chemistry.chemical_classification ,Binding Sites ,biology ,Monophenol Monooxygenase ,010405 organic chemistry ,Chemistry ,biology.organism_classification ,Recombinant Proteins ,Dihydroxyphenylalanine ,0104 chemical sciences ,Quinone ,Enzyme Activation ,030104 developmental biology ,Enzyme ,Amino Acid Substitution ,Solubility ,Reducing Agents ,Mutation ,Tyrosine ,Protein Multimerization ,Carrier Proteins ,Oxidation-Reduction ,Copper - Abstract
Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated as a complex with a “caddie” protein that assists the transport of two copper ions into the active center. In this complex, the Tyr98 residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr98 residue was converted to a reactive quinone, and resonance Raman spectroscopic a...
- Published
- 2017
19. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells
- Author
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Caroline C. Philpott, Avery G. Frey, Sarju J. Patel, and Moon-Suhn Ryu
- Subjects
Iron-Sulfur Proteins ,Models, Molecular ,0301 basic medicine ,Iron ,Nuclear Receptor Coactivators ,Ferroportin ,Biology ,Models, Biological ,Biochemistry ,Heterogeneous-Nuclear Ribonucleoproteins ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Cytosol ,Glutaredoxin ,Autophagy ,Metalloprotein ,Animals ,Humans ,Cation Transport Proteins ,Molecular Biology ,Heme ,Erythroid Precursor Cells ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Proteins ,RNA-Binding Proteins ,Minireviews ,Cell Biology ,Cell biology ,DNA-Binding Proteins ,Ferritin ,Protein Transport ,030104 developmental biology ,chemistry ,Apoferritins ,Ferritins ,biology.protein ,Chaperone complex ,Protein Multimerization ,Carrier Proteins ,Dimerization ,Molecular Chaperones - Abstract
Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells.
- Published
- 2017
20. Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation
- Author
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Omer Kabil, Janet L. Smith, Ruma Banerjee, Nicole Motl, and Meredith A. Skiba
- Subjects
Models, Molecular ,0301 basic medicine ,Sulfide ,Protein Conformation ,Mutant Chimeric Proteins ,Thiosulfates ,Sulfur metabolism ,Sulfurtransferase ,chemistry.chemical_element ,Rhodanese ,Crystallography, X-Ray ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Bacterial Proteins ,Sulfur assimilation ,Catalytic Domain ,Enzyme Stability ,Cysteine ,Disulfides ,Hydrogen Sulfide ,Quinone Reductases ,Molecular Biology ,Thiosulfate ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Burkholderiaceae ,Computational Biology ,Cell Biology ,Glutathione ,Sulfur ,Peptide Fragments ,Recombinant Proteins ,Thiosulfate Sulfurtransferase ,digestive system diseases ,030104 developmental biology ,Amino Acid Substitution ,chemistry ,Mutation ,Enzymology ,Biocatalysis ,Thiosulfate sulfurtransferase - Abstract
Hydrogen sulfide (H2S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H2S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO–rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans. The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.
- Published
- 2017
21. Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation
- Author
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Diep M.N. Nguyen, Gerrit J. Schut, John P. Hoben, Michael W. Ratzloff, Karl W. Hempel, Carolyn E. Lubner, Michael W. W. Adams, Paul W. King, and Anne-Frances Miller
- Subjects
Models, Molecular ,0301 basic medicine ,Semiquinone ,Flavodoxin ,Recombinant Fusion Proteins ,Flavoprotein ,Flavin group ,Bioenergetics ,Photochemistry ,Biochemistry ,Redox ,Electron Transport ,03 medical and health sciences ,Electron transfer ,Apoenzymes ,Bacterial Proteins ,Multienzyme Complexes ,Oxidoreductase ,Enterobacter cloacae ,Ultrafast laser spectroscopy ,NADH, NADPH Oxidoreductases ,ortho-Aminobenzoates ,Desulfovibrio vulgaris ,Molecular Biology ,Silent Mutation ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Thermus thermophilus ,Cell Biology ,Benzoic Acid ,Nitroreductases ,Recombinant Proteins ,Pyrococcus furiosus ,030104 developmental biology ,Biocatalysis ,Flavin-Adenine Dinucleotide ,biology.protein ,Holoenzymes ,Oxidoreductases ,Oxidation-Reduction - Abstract
Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to “bifurcation.” It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I and can be an indication of capacity for electron bifurcation.
- Published
- 2017
22. A Single Outer-Sphere Mutation Stabilizes apo-Mn Superoxide Dismutase by 35 °C and Disfavors Mn Binding
- Author
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Ting Wang and Anne-Frances Miller
- Subjects
0301 basic medicine ,Coordination sphere ,Stereochemistry ,Iron ,Mutant ,Mutation, Missense ,medicine.disease_cause ,Biochemistry ,Article ,Superoxide dismutase ,03 medical and health sciences ,Apoenzymes ,Enzyme Stability ,Escherichia coli ,Side chain ,medicine ,Humans ,Guanidine ,Manganese ,Mutation ,biology ,Superoxide Dismutase ,Chemistry ,Escherichia coli Proteins ,Active site ,030104 developmental biology ,Amino Acid Substitution ,Outer sphere electron transfer ,biology.protein - Abstract
The catalytic active site of Mn-specific SOD (MnSOD) is organized around a redox-active Mn ion. The most highly-conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the two types of SODs’ active sites. Mutation of this Gln69 to Glu in E. coli FeSOD increased the Fe3+/2+ reduction midpoint potential by > 0.6 V without disrupting the structure or Fe binding [E. Yikilmaz, D. W. Rodgers and A.-F. Miller (2006) Biochemistry 45(4) 1151–1161]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, the Q146E-apoMnSOD displays an apparent melting midpoint temperature (Tm) 35 °C higher that of WT-apoMnSOD, whereas the Tm of WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, the Tm attributed to Q146E-holoMnSOD is 40 °C lower than that of Q146E-apoMnSOD. Thus our data refute the notion that the WT residues optimize structural stability of the protein, being instead consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy for favoring metal ion binding.
- Published
- 2017
23. Molecular impact of covalent modifications on nonribosomal peptide synthetase carrier protein communication
- Author
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David J. Meyers, Dominique P. Frueh, and Andrew C. Goodrich
- Subjects
Models, Molecular ,0301 basic medicine ,Protein Conformation ,Yersinia pestis ,Allosteric regulation ,Fluorescence Polarization ,Calorimetry ,Biology ,Biochemistry ,Protein–protein interaction ,03 medical and health sciences ,Apoenzymes ,Bacterial Proteins ,Nonribosomal peptide ,Coenzyme A Ligases ,Protein Interaction Domains and Motifs ,Peptide Synthases ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Adenylylation ,chemistry.chemical_classification ,Carbon Isotopes ,DNA ligase ,Nitrogen Isotopes ,Protein dynamics ,Titrimetry ,Isothermal titration calorimetry ,Cell Biology ,Recombinant Proteins ,Kinetics ,030104 developmental biology ,Amino Acid Substitution ,chemistry ,Structural biology ,Mutation ,Protein Structure and Folding ,Biophysics ,Apoproteins ,Carrier Proteins ,Holoenzymes ,Protein Processing, Post-Translational ,Protein Modification, Translational - Abstract
Nonribosomal peptide synthesis involves the interplay between covalent protein modifications, conformational fluctuations, catalysis, and transient protein-protein interactions. Delineating the mechanisms involved in orchestrating these various processes will deepen our understanding of domain-domain communication in nonribosomal peptide synthetases (NRPSs) and lay the groundwork for the rational reengineering of NRPSs by swapping domains handling different substrates to generate novel natural products. Although many structural and biochemical studies of NRPSs exist, few studies have focused on the energetics and dynamics governing the interactions in these systems. Here, we present detailed binding studies of an adenylation domain and its partner carrier protein in apo-, holo-, and substrate-loaded forms. Results from fluorescence anisotropy, isothermal titration calorimetry, and NMR titrations indicated that covalent modifications to a carrier protein modulate domain communication, suggesting that chemical modifications to carrier proteins during NRPS synthesis may impart directionality to sequential NRPS domain interactions. Comparison of the structure and dynamics of an apo-aryl carrier protein with those of its modified forms revealed structural fluctuations induced by post-translational modifications and mediated by modulations of protein dynamics. The results provide a comprehensive molecular description of a carrier protein throughout its life cycle and demonstrate how a network of dynamic residues can propagate the molecular impact of chemical modifications throughout a protein and influence its affinity toward partner domains.
- Published
- 2017
24. Structural basis for substrate binding and catalytic mechanism of a human RNA:m5C methyltransferase NSun6
- Author
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Hao Li, Jing Li, En-Duo Wang, Ru-Juan Liu, and Tao Long
- Subjects
0301 basic medicine ,Models, Molecular ,Protein Conformation, alpha-Helical ,Methyltransferase ,Biology ,Bioinformatics ,Crystallography, X-Ray ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,RNA, Transfer ,Catalytic Domain ,Translational regulation ,Genetics ,Transferase ,Humans ,Amino Acid Sequence ,Conserved Sequence ,tRNA Methyltransferases ,Nucleic Acid Enzymes ,RNA ,Hydrogen Bonding ,Methylation ,TRNA Methyltransferases ,030104 developmental biology ,chemistry ,Biochemistry ,Transfer RNA ,Biocatalysis ,Cytosine ,Protein Binding - Abstract
5-methylcytosine (m5C) modifications of RNA are ubiquitous in nature and play important roles in many biological processes such as protein translational regulation, RNA processing and stress response. Aberrant expressions of RNA:m5C methyltransferases are closely associated with various human diseases including cancers. However, no structural information for RNA-bound RNA:m5C methyltransferase was available until now, hindering elucidation of the catalytic mechanism behind RNA:m5C methylation. Here, we have solved the structures of NSun6, a human tRNA:m5C methyltransferase, in the apo form and in complex with a full-length tRNA substrate. These structures show a non-canonical conformation of the bound tRNA, rendering the base moiety of the target cytosine accessible to the enzyme for methylation. Further biochemical assays reveal the critical, but distinct, roles of two conserved cysteine residues for the RNA:m5C methylation. Collectively, for the first time, we have solved the complex structure of a RNA:m5C methyltransferase and addressed the catalytic mechanism of the RNA:m5C methyltransferase family, which may allow for structure-based drug design toward RNA:m5C methyltransferase–related diseases.
- Published
- 2017
25. Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase
- Author
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Barbara Cellini, Carlotta Zamparelli, Riccardo Montioli, and Carla Borri Voltattorni
- Subjects
0301 basic medicine ,Coenzyme ,Hot Temperature ,Stereochemistry ,Dimer ,Tetramer–dimer equilibrium ,Mutation, Missense ,Bioengineering ,Biochemistry ,Article ,Cofactor ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Enzyme Stability ,Protein stability ,Humans ,Quaternary structure ,Protein Structure, Quaternary ,Pyridoxal 5′-phosphate ,Pyridoxal ,coenzyme ,interface contacts ,protein stability ,pyridoxal 5′-phosphate ,quaternary structure ,tetramer–dimer equilibrium ,Interface contacts ,Organic Chemistry ,Ornithine-Oxo-Acid Transaminase ,030102 biochemistry & molecular biology ,biology ,Ornithine ,Protein tertiary structure ,Dissociation constant ,030104 developmental biology ,Amino Acid Substitution ,chemistry ,biology.protein ,Protein quaternary structure ,Protein Multimerization ,Holoenzymes ,Intracellular - Abstract
Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent Tm values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer–dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, Tm values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s10930-017-9710-5) contains supplementary material, which is available to authorized users.
- Published
- 2017
26. Anaerobic Heme Degradation: ChuY Is an Anaerobilin Reductase That Exhibits Kinetic Cooperativity
- Author
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William N. Lanzilotta, Michael Delrossi, Joseph W. LaMattina, David B. Nix, Katherine G. Uy, Anudeep R. Neelam, and Nicholas D. Keul
- Subjects
Models, Molecular ,0301 basic medicine ,Oxidoreductases Acting on CH-CH Group Donors ,Protein Conformation ,Operon ,Stereochemistry ,Cooperativity ,Heme ,Reductase ,Escherichia coli O157 ,Biochemistry ,Article ,Cofactor ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Protein Interaction Domains and Motifs ,Molecular Structure ,030102 biochemistry & molecular biology ,biology ,Escherichia coli Proteins ,Hydrolysis ,Biliverdin reductase ,Deuterium ,Tetrapyrrole ,Porphyrin ,Recombinant Proteins ,Molecular Weight ,030104 developmental biology ,Tetrapyrroles ,chemistry ,Structural Homology, Protein ,Biocatalysis ,biology.protein ,Dimerization ,Oxidation-Reduction ,NADP - Abstract
Heme catabolism is an important biochemical process that many bacterial pathogens utilize to acquire iron. However, tetrapyrrole catabolites can be reactive and often require further processing for transport out of the cell or conversion to another useful cofactor. In previous work, we presented in vitro evidence of an anaerobic heme degradation pathway in Escherichia coli O157:H7. Consistent with reactions that have been reported for other radical S-adenosyl-L-methionine methyltransferases, ChuW transfers a methyl group to heme by a radical-mediated mechanism and catalyzes the β-scission of the porphyrin macrocycle. This facilitates iron release and the production of a new linear tetrapyrrole termed “anaerobilin”. In this work, we describe the structure and function of ChuY, an enzyme expressed downstream from chuW within the same heme utilization operon. ChuY is structurally similar to biliverdin reductase and forms a dimeric complex in solution that reduces anaerobilin to the product we have termed anaerorubin. Steady state analysis of ChuY exhibits kinetic cooperativity that is best explained by a random addition mechanism with a kinetically preferred path for initial reduced nicotinamide adenine dinucleotide phosphate binding.
- Published
- 2017
27. Activation of carbonic anhydrase IX by alternatively spliced tissue factor under late-stage tumor conditions
- Author
-
Georg F. Weber, Divya Ramchandani, Dusten Unruh, Clayton S. Lewis, and Vladimir Y. Bogdanov
- Subjects
cell division ,0301 basic medicine ,migration ,Apoenzymes ,0302 clinical medicine ,Cell Movement ,Carbonic Anhydrase Inhibitors ,Sulfonamides ,Chemistry ,Cell cycle ,Recombinant Proteins ,Neoplasm Proteins ,Enzyme Induction ,030220 oncology & carcinogenesis ,anchorage independence ,Carcinoma, Pancreatic Ductal ,G2 Phase ,Mice, Nude ,Antineoplastic Agents ,Article ,Thromboplastin ,Pathology and Forensic Medicine ,03 medical and health sciences ,Tissue factor ,Downregulation and upregulation ,Antigens, Neoplasm ,Cell Line, Tumor ,Pancreatic cancer ,medicine ,Animals ,Humans ,Carbonic Anhydrase IX ,Molecular Biology ,Cell Proliferation ,Neoplasm Staging ,Tumor microenvironment ,Tumor hypoxia ,hypoxia ,Cell growth ,Phenylurea Compounds ,Cell Biology ,tissue factor ,medicine.disease ,Xenograft Model Antitumor Assays ,cancer progression ,Pancreatic Neoplasms ,Alternative Splicing ,030104 developmental biology ,Cell culture ,Cancer research ,Tumor Hypoxia - Abstract
Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.
- Published
- 2016
28. Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils
- Author
-
Ulrich Weininger, Sven Kjellström, Shashank Deep, Mohammad Ashhar Iqbal Khan, and Mikael Akke
- Subjects
Amyloid ,Surface Properties ,SOD1 ,Nucleation ,Bioengineering ,Protein aggregation ,010402 general chemistry ,Fibril ,01 natural sciences ,Biochemistry ,Physical Chemistry ,Superoxide dismutase ,03 medical and health sciences ,Protein Aggregates ,Adsorption ,Apoenzymes ,Superoxide Dismutase-1 ,Structural Biology ,Disulfides ,Molecular Biology ,030304 developmental biology ,Protein Unfolding ,0303 health sciences ,biology ,Chemistry ,Biochemistry and Molecular Biology ,0104 chemical sciences ,Structural biology ,Biophysics ,biology.protein ,Biocatalysis ,Hydrophobic and Hydrophilic Interactions ,Intracellular ,Biotechnology - Abstract
Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.
- Published
- 2019
29. Structures of a RAG-like transposase during cut-and-paste transposition
- Author
-
David G. Schatz, Yang Yang, and Chang Liu
- Subjects
Transposable element ,Models, Molecular ,Protein domain ,Transib ,Transposases ,cryo-electron microscopy ,Moths ,Cleavage (embryo) ,Crystallography, X-Ray ,DNA-binding protein ,Article ,DNA transposition ,Evolution, Molecular ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Apoenzymes ,Protein Domains ,evolution ,Recombinase ,strand transfer ,Animals ,Amino Acid Sequence ,DNA Cleavage ,Transposase ,X-ray crystallography ,030304 developmental biology ,Homeodomain Proteins ,0303 health sciences ,Multidisciplinary ,biology ,Base Sequence ,Chemistry ,Cryoelectron Microscopy ,Active site ,DNA ,RAG ,V(D)J recombination ,Cell biology ,DNA-Binding Proteins ,biology.protein ,Biocatalysis ,030217 neurology & neurosurgery - Abstract
Transposons have had a pivotal role in genome evolution1 and are believed to be the evolutionary progenitors of the RAG1–RAG2 recombinase2, an essential component of the adaptive immune system in jawed vertebrates3. Here we report one crystal structure and five cryo-electron microscopy structures of Transib4,5, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0–4.6 A. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the ‘wings’ of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase. Analysis of multiple structures of the Helicoverpa zea DNA transposase Transib, determined by X-ray crystallography and cryo-electron microscopy, reveals the detailed pathway of the transposition reaction and sheds light on the evolution of the RAG recombinase.
- Published
- 2019
30. Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures
- Author
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Thomas M. Frimurer, Michael Toft Overgaard, Prafull S. Gandhi, Ole Hvilsted Olsen, Jesper J. Madsen, Egon Persson, and Anders B. Sorensen
- Subjects
Models, Molecular ,Protein Folding ,Proteases ,medicine.medical_treatment ,Allosteric regulation ,Biophysics ,Factor VIIa ,Crystallography, X-Ray ,Benzamidine ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,0302 clinical medicine ,Allosteric Regulation ,Protein Domains ,medicine ,Trypsin ,Disulfides ,030304 developmental biology ,0303 health sciences ,Cofactor binding ,Protease ,biology ,Chemistry ,Active site ,Articles ,Benzamidines ,Enzyme Activation ,Crystallography ,Trypsinogen ,biology.protein ,Protein folding ,Oxyanion hole ,030217 neurology & neurosurgery - Abstract
A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.
- Published
- 2019
31. Structural insight into arenavirus replication machinery
- Author
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Yi Shi, Min Wang, George F. Gao, Qi Peng, Peiyi Wang, Xichen Bao, Ruchao Peng, Sheng Liu, Xin Xu, Jianxun Qi, Jia-mei Jing, and Ying Wu
- Subjects
Models, Molecular ,Viral protein ,viruses ,Lymphocytic choriomeningitis ,medicine.disease_cause ,Virus Replication ,Virus ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Transcription (biology) ,RNA polymerase ,Catalytic Domain ,medicine ,Lymphocytic choriomeningitis virus ,Lassa virus ,Promoter Regions, Genetic ,Polymerase ,Arenaviruses, New World ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Arenavirus ,biology ,030306 microbiology ,Cryoelectron Microscopy ,virus diseases ,biology.organism_classification ,medicine.disease ,RNA-Dependent RNA Polymerase ,Virology ,Viral replication ,chemistry ,biology.protein - Abstract
Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1–4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5′-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics. The authors provide high-resolution structures of two arenavirus polymerases, revealing that the active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5′-viral RNA, and that dimerization facilitates polymerase activity.
- Published
- 2019
32. Closure of the human TKFC active site: comparison of the apoenzyme and the complexes formed with either triokinase or FMN cyclase substrates
- Author
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Joaquim Rui Rodrigues, João Meireles Ribeiro, Alicia Cabezas, and José Carlos Cameselle
- Subjects
Flavin Mononucleotide ,Flavin mononucleotide ,dihydroxyacetone kinase ,Protein domain mobility ,Substrate Specificity ,lcsh:Chemistry ,chemistry.chemical_compound ,Adenosine Triphosphate ,Apoenzymes ,Catalytic Domain ,Hydroxymethyl ,Phosphorylation ,Dihydroxyacetone kinase ,lcsh:QH301-705.5 ,Spectroscopy ,Flavin adenine dinucleotide ,biology ,General Medicine ,Computer Science Applications ,phosphoryl transfer mechanism ,Phosphoryl transfer mechanism ,Phosphotransferases (Alcohol Group Acceptor) ,molecular dynamics simulation ,Dihydroxyacetone ,Flavin-Adenine Dinucleotide ,Phosphorus-Oxygen Lyases ,Essential dynamics ,essential dynamics ,Stereochemistry ,Glyceraldehyde ,Cyclase ,Catalysis ,Article ,Inorganic Chemistry ,Molecular dynamics simulation ,Humans ,Physical and Theoretical Chemistry ,Kinase activity ,Molecular Biology ,active-site closure ,protein domain mobility ,Binding Sites ,Organic Chemistry ,Active site ,Normal mode analysis ,FMN cyclase ,triokinase ,chemistry ,lcsh:Biology (General) ,lcsh:QD1-999 ,Active-site closure ,normal mode analysis ,biology.protein ,Adenosine triphosphate ,Triokinase - Abstract
Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (&asymp, 14 Å, ) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å, or 4.16 Å, ). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP &gamma, phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.
- Published
- 2019
33. Investigation of the role of central metal ion of Cyathus bulleri laccase 1 using guanidinium chloride-induced denaturation
- Author
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Danish Idrees, Mohd. Amir, Saroj Mishra, Md. Imtaiyaz Hassan, Radhika Khera, and Sumbul Afreen
- Subjects
Guanidinium chloride ,Protein Denaturation ,Kinetics ,chemistry.chemical_element ,02 engineering and technology ,Biochemistry ,Protein Refolding ,Protein Structure, Secondary ,Pichia pastoris ,Metal ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Structural Biology ,Denaturation (biochemistry) ,Molecular Biology ,Guanidine ,030304 developmental biology ,Laccase ,0303 health sciences ,biology ,Dose-Response Relationship, Drug ,Chemistry ,General Medicine ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Copper ,Enzyme assay ,Cyathus ,Crystallography ,Zinc ,visual_art ,visual_art.visual_art_medium ,biology.protein ,Biocatalysis ,Thermodynamics ,0210 nano-technology - Abstract
The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major β-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.
- Published
- 2019
34. Evaluating apoenzyme–coenzyme–substrate interactions of methane monooxygenase with an engineered active site for electron harvesting: a computational study
- Author
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Sandun Fernando, Sikai Zhang, and Raghupathy Karthikeyan
- Subjects
Methane monooxygenase ,Coenzymes ,02 engineering and technology ,Molecular Dynamics Simulation ,Protein Engineering ,010402 general chemistry ,01 natural sciences ,Catalysis ,Cofactor ,Substrate Specificity ,Electron Transport ,Inorganic Chemistry ,chemistry.chemical_compound ,Apoenzymes ,Bacterial Proteins ,Protein Domains ,Catalytic Domain ,Physical and Theoretical Chemistry ,Flavin adenine dinucleotide ,biology ,Organic Chemistry ,Computational Biology ,Reproducibility of Results ,Active site ,021001 nanoscience & nanotechnology ,Electron transport chain ,Combinatorial chemistry ,0104 chemical sciences ,Computer Science Applications ,Molecular Docking Simulation ,Methylococcus capsulatus ,Computational Theory and Mathematics ,chemistry ,Docking (molecular) ,Anaerobic oxidation of methane ,Biocatalysis ,Oxygenases ,biology.protein ,NAD+ kinase ,0210 nano-technology ,Methane ,Protein Binding - Abstract
Low-temperature methane oxidation is one of the greatest challenges in energy research. Although methane monooxygenase (MMO) does this catalysis naturally, how to use this biocatalyst in a fuel cell environment where the electrons generated during the oxidation process is harvested and used for energy generation has not yet been investigated. A key requirement to use this enzyme in a fuel cell is wiring of the active site of the enzyme directly to the supporting electrode. In soluble MMO (sMMO), two cofactors, i.e., nicotinamide adenine di-nucleotide (NAD+) and flavin adenine dinucleotide (FAD) provide opportunities for direct attachment of the enzyme system to a supporting electrode. However, once modified to be compatible with a supporting metal electrode via FeS functionalization, how the two cofactors respond to complex binding phenomena is not yet understood. Using docking and molecular dynamic simulations, modified cofactors interactions with sMMO-reductase (sMMOR) were studied. Studies revealed that FAD modification with FeS did not interfere with binding phenomena. In fact, FeS introduction significantly improved the binding affinity of FAD and NAD+ on sMMOR. The simulations revealed a clear thermodynamically more favorable electron transport path for the enzyme system. This system can be used as a fuel cell and we can use FeS-modified-FAD as the anchoring molecule as opposed to using NAD+. The overall analysis suggests the strong possibility of building a fuel cell that could catalyze methane oxidation using sMMO as the anode biocatalyst.
- Published
- 2018
35. Enhancement of photoassembly of the functionally active water-oxidizing complex in Mn-depleted photosystem II membranes upon transition to anaerobic conditions
- Author
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Vyacheslav V. Klimov, A. A. Khorobrykh, and D. V. Yanykin
- Subjects
0301 basic medicine ,Photoinhibition ,Photosystem II ,Biophysics ,chemistry.chemical_element ,macromolecular substances ,Manganese ,Photochemistry ,Oxygen ,Cell membrane ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Spinacia oleracea ,Oxidizing agent ,medicine ,Radiology, Nuclear Medicine and imaging ,Anaerobiosis ,Radiation ,030102 biochemistry & molecular biology ,Radiological and Ultrasound Technology ,Cell Membrane ,Photosystem II Protein Complex ,Water ,food and beverages ,Membrane ,medicine.anatomical_structure ,chemistry ,Ferricyanide ,Oxidation-Reduction - Abstract
It has been shown earlier (Khorobrykh and Klimov, 2015) that molecular oxygen is directly involved in the general mechanism of the donor side photoinhibition of photosystem II (PSII) membranes. In the present work the effect of oxygen on photoassembly ("photoactivation") of the functionally active inorganic core of the water-oxidizing complex (WOC) in Mn-depleted PSII preparations (apo-WOC-PSII) in the presence of exogenous Mn(2+), Ca(2+) as well as ferricyanide was investigated. It was revealed that the efficiency of the photoassembly of the WOC was considerably increased upon removal of oxygen from the medium during photoactivation procedure using the enzymatic oxygen trap or argon flow. The lowering of O2 concentration from 250μM to 75μM, 10μM and near 0μM results in 29%, 71% and 92%, respectively, stimulation of the rate of O2 evolution measured after the photoactivation. The increase in the intensity of light used during the photoactivation was accompanied by a decrease of both the efficiency of photoassembly of the WOC and the stimulation effect of removal of O2 (that may be due to the enhancement of the processes leading to the photodamage to PSII). It is concluded that the enhancement in photoactivation of oxygen-evolving activity of apo-WOC-PSII induced by oxygen removal from the medium is due to the suppression of the donor side photoinhibition of PSII in which molecular oxygen can be involved.
- Published
- 2016
36. Chaperones in maturation of molybdoenzymes: Why specific is better than general?
- Author
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Vincent Méjean, Olivier Genest, Chantal Iobbi-Nivol, Olivier N. Lemaire, Sophie Bouillet, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)
- Subjects
0301 basic medicine ,molybdenum cofactor ,Coenzymes ,Bioengineering ,Biology ,pyranopterin monophosphate ,Models, Biological ,Applied Microbiology and Biotechnology ,MESH: general chaperone ,maturation ,molybdoenzymes ,specific chaperone ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Commentaries ,Metalloproteins ,Maturation process ,general chaperone ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,030102 biochemistry & molecular biology ,Pteridines ,Molybdoenzymes ,General Medicine ,Pterins ,Cell biology ,Co-chaperone ,030104 developmental biology ,chemistry ,Molybdenum cofactor ,Molybdenum Cofactors ,Molecular Chaperones ,Biotechnology - Abstract
International audience; Molybdoenzymes play essential functions in living organisms and, as a result, in various geochemical cycles. It is thus crucial to understand how these complex proteins become highly efficient enzymes able to perform a wide range of catalytic activities. It has been established that specific chaperones are involved during their maturation process. Here, we raise the question of the involvement of general chaperones acting in concert with dedicated chaperones or not.
- Published
- 2016
37. Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor
- Author
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Marzena Krzek, Hjalmar P. Permentier, Hugo L. van Beek, Rainer Bischoff, Marco W. Fraaije, Biotechnology, Analytical Biochemistry, and Medicinal Chemistry and Bioanalysis (MCB)
- Subjects
Models, Molecular ,Hot Temperature ,THERMOBIFIDA-FUSCA ,Protein Conformation ,Coenzymes ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,Mixed Function Oxygenases ,Sepharose ,chemistry.chemical_compound ,Apoenzymes ,TOOL ,NADH, NADPH Oxidoreductases ,heterocyclic compounds ,Monooxygenase ,Flavin adenine dinucleotide ,biology ,Protein Stability ,Microspheres ,Actinobacteria ,ESCHERICHIA-COLI ,Apo enzyme ,Flavin-Adenine Dinucleotide ,BAEYER-VILLIGER MONOOXYGENASES ,Agarose ,ENZYMES ,Biotechnology ,Immobilized enzyme ,Stereochemistry ,Recombinant Fusion Proteins ,Flavoprotein ,Bioengineering ,Flavin group ,OXIDASE ,010402 general chemistry ,Cofactor ,Immobilization ,Bacterial Proteins ,Phenylacetone monooxygenase ,Flavoenzyme ,010405 organic chemistry ,FAD ,Enzymes, Immobilized ,0104 chemical sciences ,RECONSTITUTION ,chemistry ,Biocatalysis ,biology.protein ,PHENYLACETONE MONOOXYGENASE ,BIOCATALYSTS - Abstract
A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 degrees C) when compared to PAMO in solution (no activity detected after 1 h at 60 degrees C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier. (C) 2015 Elsevier Inc. All rights reserved.
- Published
- 2016
38. Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase
- Author
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Stefanya Velásquez Gómez, Ronaldo Alves Pinto Nagem, Mozart S. Pereira, Simara Semíramis de Araújo, Denize C. Favaro, Débora Maria Abrantes Costa, Alvan C. Hengge, Tiago A. S. Brandão, Rosemeire B. Alves, and Elsevier
- Subjects
Models, Molecular ,Salicylate hydroxylase ,Decarboxylation ,Stereochemistry ,Dinitrocresols ,Catechols ,02 engineering and technology ,Flavin group ,Oxidative decarboxylation ,NahG ,Biochemistry ,Mixed Function Oxygenases ,Hydroxylation ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Structural Biology ,Catalytic Domain ,Enzyme kinetics ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,Pseudomonas putida ,Crystal structure ,General Medicine ,Monooxygenase ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Kinetics ,FAD binding ,Biocatalysis ,Thermodynamics ,Pseudomonas putida G7 ,Mechanism ,0210 nano-technology ,Salicylic Acid - Abstract
Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 A resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M−1 s−1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.
- Published
- 2018
39. Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae
- Author
-
Tsunehiro Mizushima, Kazuya Nishio, and Shu Moriyama
- Subjects
0301 basic medicine ,Models, Molecular ,Oxaloacetic Acid ,Protein Conformation, alpha-Helical ,Malates ,Gene Expression ,Nicotinamide adenine dinucleotide ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,Research Communications ,chemistry.chemical_compound ,Apoenzymes ,Structural Biology ,Malate Dehydrogenase ,Catalytic Domain ,Citrate synthase ,Cloning, Molecular ,Ternary complex ,chemistry.chemical_classification ,biology ,Chemistry ,Condensed Matter Physics ,Recombinant Proteins ,Isoenzymes ,Protein Binding ,Saccharomyces cerevisiae Proteins ,Genetic Vectors ,Biophysics ,Glyoxylate cycle ,Saccharomyces cerevisiae ,Malate dehydrogenase ,03 medical and health sciences ,Oxidoreductase ,Genetics ,Escherichia coli ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,030102 biochemistry & molecular biology ,Sequence Homology, Amino Acid ,NAD ,Citric acid cycle ,030104 developmental biology ,Glyoxysomes ,biology.protein ,Protein Conformation, beta-Strand ,NAD+ kinase ,Protein Multimerization ,Sequence Alignment - Abstract
Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported inSaccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid β-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3–NAD+complex and the MDH3–NAD+–OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.
- Published
- 2018
40. Resonance assignments for the apo-form of the cellulose-active lytic polysaccharide monooxygenase TaLPMO9A
- Author
-
Tamo Fukamizo, Yoshihito Kitaoku, Finn Lillelund Aachmann, Gaston Courtade, Dejan M. Petrović, and Vincent G. H. Eijsink
- Subjects
0301 basic medicine ,010402 general chemistry ,Polysaccharide ,01 natural sciences ,Biochemistry ,Pichia pastoris ,Mixed Function Oxygenases ,03 medical and health sciences ,chemistry.chemical_compound ,Apoenzymes ,Structural Biology ,Cellulose ,Protein secondary structure ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,biology ,Chemical shift ,Resonance ,Monooxygenase ,biology.organism_classification ,0104 chemical sciences ,030104 developmental biology ,chemistry ,Lytic cycle ,Thermoascus - Abstract
The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts. This is a post-peer-review, pre-copyedit version of an article published in [Biomolecular NMR Assignments] Locked until 16.8.2019 due to copyright restrictions. The final authenticated version is available online at: https://doi.org/10.1007/s12104-018-9839-y
- Published
- 2018
41. Transitions in DNA polymerase β μs-ms dynamics related to substrate binding and catalysis
- Author
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Samuel H. Wilson, Robert E. London, Geoffrey A. Mueller, Thomas W. Kirby, William A. Beard, and Eugene F. DeRose
- Subjects
0301 basic medicine ,Models, Molecular ,Time Factors ,DNA Repair ,DNA polymerase ,Protein Conformation ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Motion ,Apoenzymes ,Genetics ,Nucleotide ,Ternary complex ,Nuclear Magnetic Resonance, Biomolecular ,Polymerase ,DNA Polymerase beta ,chemistry.chemical_classification ,biology ,Nucleic Acid Enzymes ,Substrate (chemistry) ,Base excision repair ,DNA ,Lyase ,0104 chemical sciences ,Kinetics ,030104 developmental biology ,chemistry ,biology.protein ,Biophysics ,Biocatalysis ,Nucleic Acid Conformation ,Protein Binding - Abstract
DNA polymerase β (pol β) plays a central role in the DNA base excision repair pathway and also serves as an important model polymerase. Dynamic characterization of pol β from methyl-TROSY 13C-1H multiple quantum CPMG relaxation dispersion experiments of Ile and Met sidechains and previous backbone relaxation dispersion measurements, reveals transitions in μs-ms dynamics in response to highly variable substrates. Recognition of a 1-nt-gapped DNA substrate is accompanied by significant backbone and sidechain motion in the lyase domain and the DNA binding subdomain of the polymerase domain, that may help to facilitate binding of the apoenzyme to the segments of the DNA upstream and downstream from the gap. Backbone μs-ms motion largely disappears after formation of the pol β–DNA complex, giving rise to an increase in uncoupled μs-ms sidechain motion throughout the enzyme. Formation of an abortive ternary complex using a non-hydrolyzable dNTP results in sidechain motions that fit to a single exchange process localized to the catalytic subdomain, suggesting that this motion may play a role in catalysis.
- Published
- 2017
42. Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine
- Author
-
Giovanni Gotte, Alessandro Roncador, Riccardo Montioli, Carla Borri Voltattorni, Elisa Oppici, Mirco Dindo, and Barbara Cellini
- Subjects
Protein Folding ,Protein Conformation ,Biophysics ,Gene Expression ,CHO Cells ,Biology ,Biochemistry ,Pathogenic variant ,Analytical Chemistry ,chemistry.chemical_compound ,Apoenzymes ,Cricetulus ,Primary Hyperoxaluria Type 1 ,Pyridoxal 5′-phosphate ,Pyridoxine treatment ,Alanine:glyoxylate aminotransferase ,medicine ,Animals ,Humans ,Missense mutation ,Pyridoxal phosphate ,Molecular Biology ,Pyridoxal ,Alleles ,Transaminases ,Enzyme Assays ,Alanine ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,Chinese hamster ovary cell ,Glyoxylates ,Pyridoxine ,Peroxisome ,Recombinant Proteins ,Kinetics ,Enzyme ,Solubility ,chemistry ,Pyridoxal Phosphate ,Mutation ,Mutagenesis, Site-Directed ,Holoenzymes ,medicine.drug - Abstract
Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.
- Published
- 2015
43. Measurement of State-Specific Association Constants in Allosteric Sensors through Molecular Stapling and NMR
- Author
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Madoka Akimoto, Kody Moleschi, and Giuseppe Melacini
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Cyclic AMP-Dependent Protein Kinase RIalpha Subunit ,Allosteric regulation ,Sulfides ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,03 medical and health sciences ,Apoenzymes ,Colloid and Surface Chemistry ,Protein structure ,Allosteric Regulation ,Enzyme Stability ,Cyclic AMP ,Protein kinase A ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Affinities ,Protein Structure, Tertiary ,0104 chemical sciences ,Coupling (electronics) ,Enzyme ,Allosteric enzyme ,Mutation ,biology.protein - Abstract
Allostery is a ubiquitous mechanism to control biological function and arises from the coupling of inhibitory and binding equilibria. The extent of coupling reflects the inactive vs active state selectivity of the allosteric effector. Hence, dissecting allosteric determinants requires quantification of state-specific association constants. However, observed association constants are typically population-averages, reporting on overall affinities but not on allosteric coupling. Here we propose a general method to measure state-specific association constants in allosteric sensors based on three key elements, i.e., state-selective molecular stapling through disulfide bridges, competition binding saturation transfer experiments and chemical shift correlation analyses to gauge state populations. The proposed approach was applied to the prototypical cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA-RIα), for which the structures of the inactive and active states are available, as needed to design the state-selective disulfide bridges. Surprisingly, the PKA-RIα state-specific association constants are comparable to those of a structurally homologous domain with ∼10(3)-fold lower cAMP-affinity, suggesting that the affinity difference arises primarily from changes in the position of the dynamic apo inhibitory equilibrium.
- Published
- 2015
44. Deciphering the Molecular Basis of Functional Divergence in AMPylating Enzymes by Molecular Dynamics Simulations and Structure Guided Phylogeny
- Author
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Shradha Khater and Debasisa Mohanty
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Subfamily ,Amino Acid Motifs ,Molecular Dynamics Simulation ,Biology ,Biochemistry ,Conserved sequence ,chemistry.chemical_compound ,Adenosine Triphosphate ,Apoenzymes ,Protein structure ,Magnesium ,Adenylylation ,Conserved Sequence ,Heat-Shock Proteins ,Phylogeny ,Histidine ,Adenosine Monophosphate ,Protein Structure, Tertiary ,chemistry ,Helix ,Biocatalysis ,Solvents ,Biophysics ,Protein Processing, Post-Translational ,Adenosine triphosphate ,Functional divergence - Abstract
The Fic domain was recently shown to catalyze AMPylation-the transfer of AMP from ATP to hydroxyl side chains of diverse eukaryotic proteins, ranging from RhoGTPases to chaperon BiP. We have carried out a series of explicit solvent molecular dynamics (MD) simulations up to 1 μs duration on the apo, holo, and substrate/product bound IbpA Fic domain (IbpAFic2). Simulations on holo-IbpAFic2 revealed that binding of Mg(2+) to α and β phosphates is crucial for preserving catalytically important contacts involving ATP. Comparative analysis of the MD trajectories demonstrated how binding of ATP allosterically induces conformational changes in the distal switch II binding region of Fic domains thereby aiding in substrate recognition. Our simulations have also identified crucial aromatic-aromatic interactions which stabilize the orientation of the catalytic histidine for inline nucleophilic attack during AMPylation, thus providing a structural basis for the evolutionary conservation of these aromatic residue pairs in Fic domains. On the basis of analysis of interacting interface residue pairs that persist over the microsecond trajectory, we identified a tetrapeptide stretch involved in substrate recognition. The structure-based genome-wide search revealed a distinct conservation pattern for this segment in different Fic subfamilies, further supporting its proposed role in substrate recognition. In addition, combined use of simulations and phylogenetic analysis has helped in the discovery of a new subfamily of Fic proteins that harbor a conserved Lys/Arg in place of the inhibitory Glu of the regulatory helix. We propose the novel possibility of auto-enhancement of AMPylation activity in this new subfamily via the movement of regulatory helix, in contrast to auto-inhibition seen in most Fic proteins.
- Published
- 2015
45. Replacement of Ser108 inPlasmodium falciparumenolase results in weak Mg(II) binding: role of a parasite-specific pentapeptide insert in stabilizing the active conformation of the enzyme
- Author
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Gotam K. Jarori, Sneha Dutta, and Debanjan Mukherjee
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Recombinant Fusion Proteins ,Dimer ,Molecular Sequence Data ,Plasmodium falciparum ,Enolase ,Protozoan Proteins ,Biology ,Biochemistry ,Pentapeptide repeat ,law.invention ,Protein Aggregates ,chemistry.chemical_compound ,Apoenzymes ,law ,Enzyme Stability ,Serine ,Magnesium ,Amino Acid Sequence ,Site-directed mutagenesis ,Molecular Biology ,Conserved Sequence ,chemistry.chemical_classification ,Hydrogen Bonding ,Isothermal titration calorimetry ,Cell Biology ,Amino acid ,Kinetics ,Mutagenesis, Insertional ,Enzyme ,Amino Acid Substitution ,chemistry ,Phosphopyruvate Hydratase ,Mutagenesis, Site-Directed ,Recombinant DNA ,Mutant Proteins ,Oligopeptides ,Sequence Alignment - Abstract
A distinct structural feature of Plasmodium falciparum enolase (Pfeno) is the presence of a five amino acid insert -104EWGWS108- that is not found in host enolases. Its conservation among apicomplexan enolases has raised the possibility of its involvement in some important physiological function(s). Deletion of this sequence is known to lower k(cat)/K(m), increase K(a) for Mg(II) and convert dimer into monomers (Vora HK, Shaik FR, Pal-Bhowmick I, Mout R & Jarori GK (2009) Arch Biochem Biophys 485, 128-138). These authors also raised the possibility of the formation of an H-bond between Ser108 and Leu49 that could stabilize the apo-Pfeno in an active closed conformation that has high affinity for Mg(II). Here, we examined the effect of replacement of Ser108 with Gly/Ala/Thr on enzyme activity, Mg(II) binding affinity, conformational states and oligomeric structure and compared it with native recombinant Pfeno. The results obtained support the view that Ser108 is likely to be involved in the formation of certain crucial H-bonds with Leu49. The presence of these interactions can stabilize apo-Pfeno in an active closed conformation similar to that of Mg(II) bound yeast enolase. As predicted, S108G/A-Pfeno variants (where Ser108-Leu49 H-bonds are likely to be disrupted) were found to exist in an open conformation and had low affinity for Mg(II). They also required Mg(II) induced conformational changes to acquire the active closed conformational state essential for catalysis. The possible physiological relevance of apo-Pfeno being in such an active state is discussed.
- Published
- 2015
46. Crystal structures of apo-DszC and FMN-bound DszC fromRhodococcus erythropolisD-1
- Author
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Shipeng Wang, Takashi Ohshiro, Masaru Tanokura, Jun Ohtsuka, Li-Jun Guan, Yoshikazu Izumi, and Woo Cheol Lee
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Models, Molecular ,Flavin Mononucleotide ,Protein Conformation ,Operon ,Stereochemistry ,Static Electricity ,Protein Data Bank (RCSB PDB) ,Thiophenes ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Apoenzymes ,Protein structure ,Bacterial Proteins ,Oxidoreductase ,Catalytic Domain ,Rhodococcus ,Sulfur Dioxide ,Binding site ,Molecular Biology ,chemistry.chemical_classification ,Air Pollutants ,biology ,Chemistry ,Active site ,Cell Biology ,computer.file_format ,Protein Data Bank ,Biodegradation, Environmental ,Petroleum ,Genes, Bacterial ,Dibenzothiophene ,biology.protein ,Oxidoreductases ,computer ,Metabolic Networks and Pathways - Abstract
UNLABELLED The release of SO2 from petroleum products derived from crude oil, which contains sulfur compounds such as dibenzothiophene (DBT), leads to air pollution. The '4S' metabolic pathway catalyzes the sequential conversion of DBT to 2-hydroxybiphenyl via three enzymes encoded by the dsz operon in several bacterial species. DszC (DBT monooxygenase), from Rhodococcus erythropolis D-1 is involved in the first two steps of the '4S' pathway. Here, we determined the first crystal structure of FMN-bound DszC, and found that two distinct conformations occur in the loop region (residues 131-142) adjacent to the active site. On the basis of the DszC-FMN structure and the previously reported apo structures of DszC homologs, the binding site for DBT and DBT sulfoxide is proposed. DATABASE The atomic coordinates and structure factors for apo-DszC (PDB code: 3X0X) and DszC-FMN (PDB code: 3X0Y) have been deposited in the Protein Data Bank (http://www.rcsb.org).
- Published
- 2015
47. ApoHRP-based assay to measure intracellular regulatory heme
- Author
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Wafa Atamna, Marmik Brahmbhatt, Hani Atamna, Gregory A. Shanower, and Joseph M. Dhahbi
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Time Factors ,Heme binding ,Intracellular Space ,Biophysics ,Heme ,Biochemistry ,Horseradish peroxidase ,Article ,Cell Line ,Substrate Specificity ,Biomaterials ,chemistry.chemical_compound ,Apoenzymes ,Humans ,Horseradish Peroxidase ,Enzyme Assays ,Amyloid beta-Peptides ,biology ,Protoporphyrin IX ,Metals and Alloys ,Mercury ,Reference Standards ,Molecular biology ,Lead ,chemistry ,Chemistry (miscellaneous) ,Catalase ,biology.protein ,Intracellular ,Hemin ,Peroxidase - Abstract
The majority of the heme-binding proteins possess a “heme-pocket” that stably binds with heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the “Heme-Regulatory Motifs” (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independently from the total heme (TH). The current study describes and validates a new method to measure intracellular RH. The method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent from TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β(Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ~6% of total heme in IMR90 cells.
- Published
- 2015
48. Structural insight into the conformational change of alcohol dehydrogenase from Arabidopsis thaliana L. during coenzyme binding
- Author
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FangFang Chen, JianQin Huang, Ping Wang, Yan An, and Yingwu Xu
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Models, Molecular ,Conformational change ,Stereochemistry ,Arabidopsis ,Crystallography, X-Ray ,Biochemistry ,Apoenzymes ,Species Specificity ,Oxidoreductase ,Animals ,Humans ,Coenzyme binding ,Horses ,Binding site ,Protein Structure, Quaternary ,Alcohol dehydrogenase ,chemistry.chemical_classification ,Binding Sites ,biology ,Alcohol Dehydrogenase ,Active site ,General Medicine ,NAD ,NAD binding ,chemistry ,biology.protein ,NAD+ kinase ,Protein Multimerization ,Protein Binding - Abstract
Alcohol dehydrogenase (ADH, EC 1.1.1.1) plays important roles in the metabolism of alcohols and aldehydes. They are often subjected to conformational changes that are critical for the enzymatic activity and have received intensive investigation for horse liver ADH. However, for the large plant ADH members, little is known regarding both the conformational change and its relationship to catalytic activity as plant ADH structures were rarely available. Here we describe three Arabidopsis ADH conformations obtained from two crystals, the apo crystal that was free of ligand, and the complex crystal that was with NAD. The NAD-complexed crystal yielded two different structural forms for the two subunits, one was occupied by the coenzyme, and the other was free and open. Structural comparisons demonstrate that the occupied subunit is in a closed conformation while the free subunit is fully open, and the apo structure in intermediate. Though all the forms have an overall fold similar to that of horse and human ADHs, the catalytic domain has an over 10° rotation. Additionally, unlike horse liver ADH, the loop (295-302aa) adopts different conformation. It does not rearrange upon the binding of the coenzyme norVal297 side chain experiences a flipping. Instead it always remains in the active site. His48 plays a switching role in the structure. Its imidazole ring has to swim away from the binding site to permit NAD binding. These together with the large differences in the substrate binding pocket, as well as in the proton relay system demonstrate that AtADH adopts a different catalysis mechanism from horse liver ADH.
- Published
- 2015
49. Inhibition studies of DNA methyltransferases by maleimide derivatives of RG108 as non-nucleoside inhibitors
- Author
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Céline Faux, Johan Wouters, Grégoire Rondelet, Véronique Masson, Paola B. Arimondo, Jean Dubois, Laurence Fleury, Luc Willems, Université de Namur [Namur] (UNamur), Pharmacochimie de la Régulation Epigénétique du Cancer (ETaC), PIERRE FABRE-Centre National de la Recherche Scientifique (CNRS), Churchill College [Cambridge], University of Cambridge [UK] (CAM), Université de Liège, PIERRE FABRE-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-PIERRE FABRE
- Subjects
0301 basic medicine ,Methyltransferase ,[SDV]Life Sciences [q-bio] ,MESH: Recombinant Proteins ,Maleimides ,MESH: Protein Structure, Tertiary ,chemistry.chemical_compound ,Mice ,MESH: DNA Methylation ,Apoenzymes ,Drug Discovery ,MESH: Animals ,Fluorometry ,DNA (Cytosine-5-)-Methyltransferases ,Enzyme Inhibitors ,MESH: Inhibitory Concentration 50 ,MESH: Maleimides ,Chemistry ,Tryptophan ,DNA methyltransferases ,MESH: Apoenzymes ,Recombinant Proteins ,3. Good health ,Molecular Docking Simulation ,MESH: Cell Survival ,Biochemistry ,MESH: Enzyme Inhibitors ,DNA methylation ,Molecular Medicine ,Epigenetic therapy ,apoenzyme crystal structure ,MESH: DNA (Cytosine-5-)-Methyltransferases ,MESH: Cell Line, Tumor ,Cell Survival ,Phthalimides ,03 medical and health sciences ,Inhibitory Concentration 50 ,Cell Line, Tumor ,MESH: Molecular Docking Simulation ,MESH: Phthalimides ,[CHIM]Chemical Sciences ,Animals ,Humans ,Mode of action ,MESH: Mice ,MESH: Tryptophan ,Pharmacology ,MESH: Humans ,Binding Sites ,epigenetics ,MESH: Fluorometry ,DNA Methylation ,Protein Structure, Tertiary ,030104 developmental biology ,MESH: Binding Sites ,Docking (molecular) ,Cancer cell ,Michael acceptor ,Nucleoside ,DNA - Abstract
Aim: DNA methyltransferases (DNMTs) are important drug targets for epigenetic therapy of cancer. Nowadays, non-nucleoside DNMT inhibitors are in development to address high toxicity of nucleoside analogs. However, these compounds still have low activity in cancer cells and mode of action of these compounds remains unclear. Materials & methods: In this work, we studied maleimide derivatives of RG108 by biochemical, structural and computational approaches to highlight their inhibition mechanism on DNMTs. Results: Findings demonstrated a correlation between cytotoxicity on mesothelioma cells of these compounds and their inhibitory potency against DNMTs. Noncovalent and covalent docking studies, supported by crystallographic (apo structure of M.HhaI) and differential scanning fluorimetry assays, provided detailed insights into their mode of action and revealed essential residues for the stabilization of such compounds inside DNMTs. [Formula: see text]
- Published
- 2017
50. Structural and functional characterization of the Vindoline biosynthesis pathway enzymes of Catharanthus roseus
- Author
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Sudeshna Bose, Saikat Chakrabarti, Harshita Tiwari, Shrabasti Jana, Anindyajit Banerjee, and Bilal Ahmad
- Subjects
0301 basic medicine ,Models, Molecular ,Molecular model ,Catharanthus ,Vinblastine ,01 natural sciences ,Catalysis ,Mixed Function Oxygenases ,Inorganic Chemistry ,03 medical and health sciences ,Apoenzymes ,Acetyltransferases ,Catalytic Domain ,0103 physical sciences ,Homology modeling ,Physical and Theoretical Chemistry ,Plant Proteins ,chemistry.chemical_classification ,010304 chemical physics ,biology ,Chemistry ,Organic Chemistry ,Tabersonine ,Catharanthine ,Methyltransferases ,Catharanthus roseus ,biology.organism_classification ,Enzyme structure ,Computer Science Applications ,Biosynthetic Pathways ,Molecular Docking Simulation ,030104 developmental biology ,Enzyme ,Computational Theory and Mathematics ,Biochemistry ,Oxygenases ,Vindoline - Abstract
Vinblastine and its related compound vincristine are important mono terpenoid indole alkaloids accumulated in the leaves of Catharanthus roseus (Madagascar periwinkle). They serve as major anticancer drugs. Vinblastine is formed by the condensation of vindoline and catharanthine. The vindoline moiety is derived from tabersonine via vindoline biosynthesis pathway. The reaction sequence from tabersonine to vindoline is now well established and the enzymes involved in this pathway are identified. However, to date, the structures of the enzymes involved in the vindoline biosynthesis pathway are not known, leading to limited mechanistic understanding of the substrate binding and catalysis. The purpose of this work is to provide structural insight regarding all the steps of the vindoline pathway via rigorous homology modeling, molecular docking, and molecular dynamics analyses. Substrate and cofactors required for each step were docked onto the computationally built and validated three-dimensional (3D) model of the corresponding enzyme, and the catalytic reaction was analyzed from the structural point of view. Possible binding modes of the substrates and cofactors were generated and corresponding binding residues were identified. Enzyme-substrate models were verified based on structure evaluation methods and molecular dynamics based approaches. Findings of our analysis would be useful in rational designing of these important enzymes aimed toward bio-production of vindoline.
- Published
- 2017
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