4,716 results on '"lyase"'
Search Results
2. Characterization of Molecular Diversity and Organization of Phycobilisomes in Thermophilic Cyanobacteria.
- Author
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Tang, Jie, Zhou, Huizhen, Yao, Dan, Du, Lianming, and Daroch, Maurycy
- Subjects
- *
AMINO acid sequence , *PHYCOBILISOMES , *DIVERSITY in organizations , *THERMOPHILIC bacteria , *CYANOBACTERIA , *GENOMICS - Abstract
Thermophilic cyanobacteria are cosmopolitan and abundant in the thermal environment. Their light-harvesting complexes, phycobilisomes (PBS), are highly important in photosynthesis. To date, there is limited information on the PBS composition of thermophilic cyanobacteria whose habitats are challenging for survival. Herein, genome-based methods were used to investigate the molecular components of PBS in 19 well-described thermophilic cyanobacteria. These cyanobacteria are from the genera Leptolyngbya, Leptothermofonsia, Ocullathermofonsia, Thermoleptolyngbya, Trichothermofonsia, Synechococcus, Thermostichus, and Thermosynechococcus. According to the phycobiliprotein (PBP) composition of the rods, two pigment types are observed in these thermophiles. The amino acid sequence analysis of different PBP subunits suggests several highly conserved cysteine residues in these thermophiles. Certain amino acid contents in the PBP of thermophiles are significantly higher than their mesophilic counterparts, highlighting the potential roles of specific substitutions of amino acid in the adaptive thermostability of light-harvesting complexes in thermophilic cyanobacteria. Genes encoding PBS linker polypeptides vary among the thermophiles. Intriguingly, motifs in linker apcE indicate a photoacclimation of a far-red light by Leptolyngbya JSC-1, Leptothermofonsia E412, and Ocullathermofonsia A174. The composition pattern of phycobilin lyases is consistent among the thermophiles, except for Thermostichus strains that have extra homologs of cpcE, cpcF, and cpcT. In addition, phylogenetic analyses of genes coding for PBPs, linkers, and lyases suggest extensive genetic diversity among these thermophiles, which is further discussed with the domain analyses. Moreover, comparative genomic analysis suggests different genomic distributions of PBS-related genes among the thermophiles, indicating probably various regulations of expression. In summary, the comparative analysis elucidates distinct molecular components and organization of PBS in thermophilic cyanobacteria. These results provide insights into the PBS components of thermophilic cyanobacteria and fundamental knowledge for future research regarding structures, functions, and photosynthetic improvement. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
3. 微生物来源的果胶相关裂解酶的研究进展.
- Author
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郑玲, 李谦, 徐寅啸, 宁利敏, 朱本伟, and 姚忠
- Abstract
Copyright of Chinese Journal of Bioprocess Engineering is the property of Chinese Journal of Bioprocess Engineering Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
- Published
- 2022
- Full Text
- View/download PDF
4. Self-Assembled Fullerene Nanostructures for Mimicking and Understanding of Natural Enzymes.
- Author
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Demirsoy, Zeynep and Gulseren, Gulcihan
- Abstract
Self-assembling enzyme mimics offer an easy way to imitate activities of natural enzymes but have not been thus far used to understand the effect of different amino acids on the catalytic activity and why they are evolutionarily preserved for specific catalytic roles. Here, we demonstrated that fullerene nanostructures functionalized with catalytically active amino acids, which form multiple active sites via the self-assembly process in the aqueous environment, serve as an effective system to distinguish the catalytic activity differences resulting from single amino acid changes. A nano-level tuning of intermolecular and intramolecular interactions enabled formation of efficient enzyme mimics. Furthermore, using the carboxyl–imidazole couple found in quite different enzymes as the main catalytic unit, we could mimic different enzyme classes, like hydrolases and lyases, with significant catalytic activities. These designed nanocatalysts were also reusable and catalytically active under physiological conditions like natural enzymes. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
5. Cloning and Expression of Recombinant Chondroitinase ACII and Its Comparison to the Arthrobacter aurescens Enzyme.
- Author
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Williams, Asher, He, Wenqin, Cress, Brady F, Liu, Xinyue, Alexandria, Jordanne, Yoshizawa, Hiroki, Nishimura, Kazuhiro, Toida, Toshihiko, Koffas, Mattheos, and Linhardt, Robert J
- Subjects
Escherichia coli ,Arthrobacter ,Chondroitin Lyases ,Chondroitin ,Chondroitin Sulfates ,Recombinant Proteins ,Enzyme Stability ,Temperature ,Gene Expression Regulation ,Bacterial ,Protein Processing ,Post-Translational ,Enzyme Activation ,Substrate Specificity ,Point Mutation ,Genetic Vectors ,chondroitin sulfate ,chondroitinase ,lyase ,recombinant expression ,specificity ,Environmental Biotechnology ,Industrial Biotechnology ,Medical Biotechnology ,Biotechnology - Abstract
Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study is to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII are prepared in Escherichia coli, and their activity, stability, specificity, and action pattern are examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes are similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences toward uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter-secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.
- Published
- 2017
6. Isolation, Diversity and Characterization of Ulvan-Degrading Bacteria Isolated from Marine Environments.
- Author
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Tanaka, Reiji, Kurishiba, Yu, Miyake, Hideo, and Shibata, Toshiyuki
- Subjects
- *
LYASES , *GREEN algae , *VIBRIO , *ULVA , *HYDROLASES - Abstract
In this study, we aimed to isolate bacteria capable of degrading the polysaccharide ulvan from the green algae Ulva sp. (Chlorophyta, Ulvales, Ulvaceae) in marine environments. We isolated 13 ulvan-degrading bacteria and observed high diversity at the genus level. Further, the genera Paraglaciecola, Vibrio, Echinicola, and Algibacter, which can degrade ulvan, were successfully isolated for the first time from marine environments. Among the 13 isolates, only one isolate (Echinicola sp.) showed the ability not only to produce externally expressed ulvan lyase, but also to be periplasmic or on the cell surface. From the results of the full-genome analysis, lyase was presumed to be a member of the PL25 (BNR4) family of ulvan lyases, and the bacterium also contained the sequence for glycoside hydrolase (GH43, GH78 and GH88), which is characteristic of other ulvan-degrading bacteria. Notably, this bacterium has a unique ulvan lyase gene not previously reported. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
7. Investigation of the Molecular Mechanisms of the Eukaryotic Cytochrome- c Maturation System.
- Author
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Silva, Ana V., Firmino, Maria O., Costa, Nazua L., Louro, Ricardo O., and Paquete, Catarina M.
- Subjects
- *
HEMOPROTEINS , *AMINO acid sequence , *SHEWANELLA oneidensis , *VINYL polymers , *CHARGE exchange - Abstract
Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
8. A chemo-enzymatic method for preparation of saturated oligosaccharides from alginate and other uronic acid-containing polysaccharides.
- Author
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Gravdahl, Mina, Aarstad, Olav A., Petersen, Agnes B., Karlsen, Stina G., Donati, Ivan, Czjzek, Mirjam, Åstrand, Ove Alexander Høgmoen, Rye, Philip D., Tøndervik, Anne, Sletta, Håvard, Aachmann, Finn L., and Skjåk-Bræk, Gudmund
- Subjects
- *
POLYSACCHARIDES , *URONIC acids , *CHEMICAL decomposition , *ALGINIC acid , *HEPARIN , *OLIGOSACCHARIDES - Abstract
Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy- l - erythro -hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α- l -guluronate oligosaccharides with a lower content of β- d -mannuronate residues compared to what can be achieved using chemical depolymerization alone. [Display omitted] [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
9. Research progress on Streptococcus mutans phages in the prevention of dental caries
- Author
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LI Yuhan, LI Jiaxin, ZHANG Shiming, ZHANG Yaohua, LI Yuqing, and ZENG Jumei
- Subjects
streptococcus mutans ,bacteriophage ,dental caries ,prevention of dental caries ,phage therapy ,lyase ,biofilm ,cocktail therapy ,Medicine - Abstract
The Streptococcus mutans (S. mutans) phage, as one of the principal pathogenic bacteria of dental caries, is a main cause of the formation and development of dental caries due to its overproliferation in dental plaque biofilms. Bacterial viruses, also known as bacteriophages, have the capability of specifically infecting bacteria and effectively degrading bacterial biofilms. S. mutans phages, therefore, may prevent and control caries. Therapy based on phages has been applied in many fields, but the application of S. mutans phages in caries remains exploratory. This article will review the research progress of S. mutans phages in clinical caries prevention, aiming to provide a new idea for the clinical prevention of caries. The results of the literature review show that the living bacteriophage system has the advantages of high specificity, high affinity and good safety. However, due to its unstable structure, it can be processed into a more stable formulation by freeze-drying, spray drying, adding stability enhancers, or incorporating bacteriophages into ointments, biodegradable polymer matrices or particles to a certain extent to improve stability. The lysozyme produced by phages can digest the bacterial cell wall and release the assembled phage particles, which effectively cleave biofilms. In addition, the antigen binding fragment library for cariogenic pathogens was screened by phage display technology, and the purpose of caries prevention and treatment was achieved by passive immunization of antigen binding fragments. However, the host range of bacteriophages is narrow, so this kind of problem can be overcome by phage combined with traditional therapy or other drug use or cocktail therapy with multiple phages in clinical caries prevention and control.
- Published
- 2021
- Full Text
- View/download PDF
10. Characterization of Molecular Diversity and Organization of Phycobilisomes in Thermophilic Cyanobacteria
- Author
-
Jie Tang, Huizhen Zhou, Dan Yao, Lianming Du, and Maurycy Daroch
- Subjects
thermophilic cyanobacterium ,phycobiliprotein ,phycobilisome ,linker protein ,lyase ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Thermophilic cyanobacteria are cosmopolitan and abundant in the thermal environment. Their light-harvesting complexes, phycobilisomes (PBS), are highly important in photosynthesis. To date, there is limited information on the PBS composition of thermophilic cyanobacteria whose habitats are challenging for survival. Herein, genome-based methods were used to investigate the molecular components of PBS in 19 well-described thermophilic cyanobacteria. These cyanobacteria are from the genera Leptolyngbya, Leptothermofonsia, Ocullathermofonsia, Thermoleptolyngbya, Trichothermofonsia, Synechococcus, Thermostichus, and Thermosynechococcus. According to the phycobiliprotein (PBP) composition of the rods, two pigment types are observed in these thermophiles. The amino acid sequence analysis of different PBP subunits suggests several highly conserved cysteine residues in these thermophiles. Certain amino acid contents in the PBP of thermophiles are significantly higher than their mesophilic counterparts, highlighting the potential roles of specific substitutions of amino acid in the adaptive thermostability of light-harvesting complexes in thermophilic cyanobacteria. Genes encoding PBS linker polypeptides vary among the thermophiles. Intriguingly, motifs in linker apcE indicate a photoacclimation of a far-red light by Leptolyngbya JSC-1, Leptothermofonsia E412, and Ocullathermofonsia A174. The composition pattern of phycobilin lyases is consistent among the thermophiles, except for Thermostichus strains that have extra homologs of cpcE, cpcF, and cpcT. In addition, phylogenetic analyses of genes coding for PBPs, linkers, and lyases suggest extensive genetic diversity among these thermophiles, which is further discussed with the domain analyses. Moreover, comparative genomic analysis suggests different genomic distributions of PBS-related genes among the thermophiles, indicating probably various regulations of expression. In summary, the comparative analysis elucidates distinct molecular components and organization of PBS in thermophilic cyanobacteria. These results provide insights into the PBS components of thermophilic cyanobacteria and fundamental knowledge for future research regarding structures, functions, and photosynthetic improvement.
- Published
- 2023
- Full Text
- View/download PDF
11. 噬菌体裂解酶在食品安全领域的研究进展.
- Author
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孙新城, 胡旭阳, 许素月, 李侠颖, 李 爽, 周 杰, 赵成鑫, 胡金强, 高 辉, 耿 尧, 杨德良, 白艳红, and 张晓根
- Abstract
Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
- Published
- 2021
12. Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis.
- Author
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Schelch, Sabine, Eibinger, Manuel, Gross Belduma, Stefanie, Petschacher, Barbara, Kuballa, Jürgen, and Nidetzky, Bernd
- Abstract
Sialo‐oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio‐catalysis is central to the development of sialo‐oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialo‐oligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling‐guided approach to 3ʹ‐sialyllactose (3SL) synthesis from N‐acetyl‐ d‐neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5ʹ‐triphosphate, via the reactions of cytidine 5ʹ‐monophosphate‐Neu5Ac synthetase and α2,3‐sialyltransferase. The Neu5Ac was synthesized in situ from N‐acetyl‐ d‐mannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase. We show through comprehensive time‐course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model‐based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%–75%; 7–10 g/L) and overall productivity (3–5 g/L/h). Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme‐catalyzed transformations fit for oligosaccharide production. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
13. Biochemical Characterization of a Novel Thermostable Ulvan Lyase from Tamlana fucoidanivorans CW2-9.
- Author
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Xu Y, Li J, An L, Qiu Y, Mao A, He Z, Guo J, Yan H, Li H, and Hu Z
- Subjects
- Kinetics, Hot Temperature, Hydrogen-Ion Concentration, Mutagenesis, Site-Directed, Substrate Specificity, Molecular Docking Simulation, Bacterial Proteins genetics, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Ulva chemistry, Ulva enzymology, Ulva genetics, Molecular Dynamics Simulation, Polysaccharide-Lyases genetics, Polysaccharide-Lyases chemistry, Polysaccharide-Lyases metabolism, Enzyme Stability, Polysaccharides chemistry, Polysaccharides metabolism
- Abstract
Ulvan is a complex sulfated polysaccharide extracted from Ulva , and ulvan lyases can degrade ulvan through a β-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The K
m and Vmax of recombinant EPL15085 toward ulvan are 0.80 mg·mL-1 and 11.22 μmol·min-1 mg-1 ·mL-1 , respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.- Published
- 2024
- Full Text
- View/download PDF
14. The Promiscuous Activity of the Radical SAM Enzyme NosL toward Two Unnatural Substrates.
- Author
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Yin, Yue, Ji, Xinjian, and Zhang, Qi
- Subjects
- *
PEPTIDE antibiotics , *ENZYMES , *LYASES , *CARBOXYL group , *TRYPTOPHAN - Abstract
Main observation and conclusion: The radical S‐adenosylmethionine (SAM) enzyme NosL catalyzes the conversion of L‐tryptophan (L‐Trp, 1) to 3‐methyl‐2‐indolic acid (MIA, 2), a key intermediate in the biosynthesis of the peptide antibiotic nosiheptide. Previous study showed that this remarkable recombination reaction starts from the cleavage of the Cα—COO– bond to result in a •CO2− radical migration. In contrast to the radical SAM tyrosine lyases, NosL appears unable to cleave the Cα—Cβ bond, which is intrinsically more favorable to be cleaved than the Cα—COO– bond. In this study, we investigate the NosL activity with tryptamine (11) and tryptophol (12), two L‐Trp analogues lacking a carboxylate moiety. We showed that NosL cleaves the C1—C2 bond of these two substrates to produce 3‐methylindole (7), suggesting that the enzyme can still catalyze a β‐scission when the carboxyl group of Trp is absent. We also showed the enzyme exhibits a promiscuous activity, initiating the reaction by abstracting hydrogen atoms from two different sites to produce two sets of products. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
15. Targeting of Steroid Hormone Receptor Function in Breast and Prostate Cancer
- Author
-
Gupta, Shilpa, Yee, Douglas, Lenzi, Andrea, Series editor, Jannini, Emmanuele A., Series editor, Belfiore, Antonino, editor, and LeRoith, Derek, editor
- Published
- 2018
- Full Text
- View/download PDF
16. Isolation, Diversity and Characterization of Ulvan-Degrading Bacteria Isolated from Marine Environments
- Author
-
Reiji Tanaka, Yu Kurishiba, Hideo Miyake, and Toshiyuki Shibata
- Subjects
ulvan ,ulva ,degradation ,lyase ,bacteria ,paraglaciecola ,Organic chemistry ,QD241-441 - Abstract
In this study, we aimed to isolate bacteria capable of degrading the polysaccharide ulvan from the green algae Ulva sp. (Chlorophyta, Ulvales, Ulvaceae) in marine environments. We isolated 13 ulvan-degrading bacteria and observed high diversity at the genus level. Further, the genera Paraglaciecola, Vibrio, Echinicola, and Algibacter, which can degrade ulvan, were successfully isolated for the first time from marine environments. Among the 13 isolates, only one isolate (Echinicola sp.) showed the ability not only to produce externally expressed ulvan lyase, but also to be periplasmic or on the cell surface. From the results of the full-genome analysis, lyase was presumed to be a member of the PL25 (BNR4) family of ulvan lyases, and the bacterium also contained the sequence for glycoside hydrolase (GH43, GH78 and GH88), which is characteristic of other ulvan-degrading bacteria. Notably, this bacterium has a unique ulvan lyase gene not previously reported.
- Published
- 2022
- Full Text
- View/download PDF
17. Investigation of the Molecular Mechanisms of the Eukaryotic Cytochrome-c Maturation System
- Author
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Ana V. Silva, Maria O. Firmino, Nazua L. Costa, Ricardo O. Louro, and Catarina M. Paquete
- Subjects
lyase ,multiheme cytochrome ,Small Tetraheme Cytochrome ,CcHL ,Microbiology ,QR1-502 - Abstract
Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems.
- Published
- 2022
- Full Text
- View/download PDF
18. Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family
- Author
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Xiaohu Guo, Annika Söderholm, Sandesh Kanchugal P, Geir V Isaksen, Omar Warsi, Ulrich Eckhard, Silvia Trigüis, Adolf Gogoll, Jon Jerlström-Hultqvist, Johan Åqvist, Dan I Andersson, and Maria Selmer
- Subjects
bacteriophage ,S-adenosyl methionine ,lyase ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.
- Published
- 2021
- Full Text
- View/download PDF
19. Biosynthesis of a Phycocyanin Beta Subunit with Two Noncognate Chromophores in Escherichia coli.
- Author
-
Chen, Huaxin, Zheng, Caiyun, Jiang, Peng, and Ji, Gengsheng
- Abstract
Recombinant phycobiliprotein can be used as fluorescent label in immunofluorescence assay. In this study, pathway for phycocyanin beta subunit (CpcB) carrying noncognate chromophore phycoerythrobilin (PEB) and phycourobilin (PUB) was constructed in Escherichia coli. Lyase CpcS and CpcT could catalyze attachment of PEB to Cys84-CpcB and Cys155-CpcB, respectively. However, PEB was attached only to Cys84-CpcB when both CpcS and CpcT were present in E. coli. A dual plasmid expression system was used to control the expression of lyases and the attachment order of PEB to CpcB. The production of PEB-Cys155-CpcB was achieved by L-arabinose-induced expression of CpcS, CpcB, Ho1, and PebS, and then the attachment of PEB to Cys84-CpcB was achieved by IPTG-induced expression of CpcS. The doubly chromophorylated CpcB absorbed light maximally at 497.5 nm and 557.0 nm and fluoresced maximally at 507.5 nm and 566.5 nm. An amount of light energy absorbed by PUB-Cys155-CpcB is transferred to PEB-Cys84-CpcB in doubly chromophorylated CpcB, conferring a large stokes shift of 69 nm for this fluorescent protein. There are interactions between chromophores of CpcB which possibly together with the help of lyases lead to isomerization of PEB-Cys155-CpcB to PUB-Cys155-CpcB. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
20. Extending the enzymatic toolbox for heparosan polymerization, depolymerization, and detection.
- Author
-
Sulewska, Małgorzata, Berger, Monika, Damerow, Manuela, Schwarzer, David, Buettner, Falk F.R., Bethe, Andrea, Taft, Manuel H., Bakker, Hans, Mühlenhoff, Martina, Gerardy-Schahn, Rita, Priem, Bernard, and Fiebig, Timm
- Subjects
- *
ESCHERICHIA coli , *DEPOLYMERIZATION , *BIOPOLYMERS , *SITE-specific mutagenesis , *POLYMERIZATION , *HEPARIN , *POLYMERS - Abstract
Heparosan is an acidic polysaccharide expressed as a capsule polymer by pathogenic and commensal bacteria, e.g. by E. coli K5. As a precursor in the biosynthesis of heparan sulfate and heparin, heparosan has a high biocompatibility and is thus of interest for pharmaceutical applications. However, due to its low immunogenicity, developing antibodies against heparosan and detecting the polymer in biological samples has been challenging. In this study, we exploited the enzyme repertoire of E. coli K5 and the E. coli K5-specific bacteriophage ΦK5B for the controlled synthesis and depolymerization of heparosan. A fluorescently labeled heparosan nonamer was used as a priming acceptor to study the elongation mechanism of the E. coli K5 heparosan polymerases KfiA and KfiC. We could demonstrate that the enzymes act in a distributive manner, producing labeled heparosan of low dispersity. The enzymatically synthesized heparosan was a useful tool to identify the tailspike protein KflB of ΦK5B as heparosan lyase and to characterize its endolytic depolymerization mechanism. Most importantly, using site-directed mutagenesis and rational construct design, we generated an inactive version of KflB for the detection of heparosan in ELISA-based assays, on blots, and on bacterial and mammalian cells. [Display omitted] [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
21. Interplay between differentially expressed enzymes contributes to light color acclimation in marine Synechococcus.
- Author
-
Sanfilippo, Joseph E., Nguyen, Adam A., Garczarek, Laurence, Karty, Jonathan A., Pokhrel, Suman, Strnat, Johann A., Partensky, Frédéric, Schluchter, Wendy M., and Kehoe, David M.
- Subjects
- *
SYNECHOCOCCUS , *BACTERIAL enzymes , *ACCLIMATIZATION , *MARINE microbiology , *GENE expression in bacteria - Abstract
Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to the light-harvesting protein MpeA in green light, while MpeZ attaches PUB to MpeA in blue light. We demonstrate that the ratio of mpeY to mpeZ mRNA determines if PEB or PUB is attached. Additionally, strains encoding only MpeY or MpeZ do not acclimate. Examination of strains of Synechococcus isolated from across the globe indicates that the interplay between MpeY and MpeZ uncovered here is a critical feature of chromatic acclimation for marine Synechococcus worldwide. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
22. Identification and characterization of a sterically robust phenylalanine ammonia-lyase among 481 natural isoforms through association of in silico and in vitro studies.
- Author
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Rahmatabadi, Seyyed Soheil, Sadeghian, Issa, Ghasemi, Younes, Sakhteman, Amirhossein, and Hemmati, Shiva
- Subjects
- *
PHENYLALANINE ammonia lyase , *SILICON , *MOLECULAR dynamics , *MOLECULAR docking , *STATISTICAL correlation - Abstract
Graphical abstract Highlights • 481 species producing phenylalanine ammonia lyase (PAL) were selected from Uniprot. • PAL6 from Lotus japonicus (LjPAL6) was the most stable PAL in in silico analyses. • Molecular dynamic simulation and docking showed high affinity of LjPAL6 and l -Phe. • LjPAL6 showed K m and K cat values of 0.483 mM, and 7 S−1, respectively. • Overexpression and optimization of Ljpal6 resulted in 83.7 mg/l enzyme production. Abstract The enzyme phenylalanine ammonia lyase (PAL) is of special importance for the treatment of phenylketonuria patients. The aim of this study was to find a stable recombinant PAL with suitable kinetic properties among all natural PAL producing species using in silico and experimental approaches. To find such a stable PAL among 481 natural isoforms, 48,000 of 3-D models were predicted using the Modeller 9.10 program and evaluated by Ramachandran plot. Correlation analysis between Ramachandran plot and the energy of different thermodynamic components indicated that this plot could be an appropriate tool to predict protein stability. Hence, PAL6 from Lotus japonicus (LjPAL6) was selected as a stable isoform. Molecular dynamic (MD) simulation for 50 ns and docking has been conducted for LjPAL6-phenylalanine complex. The best PAL-phenylalanine frame was selected by re-docking with l -phenylalanine (L-Phe) and root-mean-square deviation (RMSD) value. MD simulation showed that the complex has a good stability, depicted by the low RMSD value, binding free energy and hydrogen bindings. Docking results showed that LjPAL6 has a high affinity toward l -Phe according to the low level of binding free energy. By overexpressing Ljpal6 in E. coli BL21, a total of 33.5 mg/l of protein was obtained, which has been increased to 83.7 mg/l via the optimization of LjPAL6 production using response surface methodology. The optimal pH and temperature were 8.5 and 50 °C, respectively. LjPAL6 showed a specific activity of 42 nkat/mg protein, with K m , K cat and K cat / K m values of 0.483 mM, 7 S−1 and 14.5 S−1 mM−1 for l -phe, respectively. In conclusion, finding models with the most reasonable stereo-chemical quality and lowest numbers of steric clashes would result in easier folding. Hence, in silico analyses of bulk data from natural origin will lead one to find an optimal model for in vitro studies and drug design. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
23. Bacterial CYP154C8 catalyzes carbon‐carbon bond cleavage in steroids.
- Author
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Dangi, Bikash and Oh, Tae‐Jin
- Subjects
- *
STEROIDS , *RING formation (Chemistry) , *PREDNISONE , *CYTOCHROME P-450 , *NADPH oxidase , *HYDROXYLATION - Abstract
Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C–C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR‐based structural elucidation of reaction products reveals 21‐hydroxyprednisone as the major product for prednisone, while the other product is identified as 1‐dehydroadrenosterone obtained due to C–C bond cleavage. A similar pattern of product formation is observed with cortisone, hydrocortisone, and prednisone. The reaction catalyzed by CYP154C8 in the presence of oxygen surrogates also prominently shows the formation of C–C bond cleavage products. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
24. Biosynthesis of a New Fusaoctaxin Virulence Factor in Fusarium graminearum Relies on a Distinct Path To Form a Guanidinoacetyl Starter Unit Priming Nonribosomal Octapeptidyl Assembly
- Author
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Hao-Yu Tang, Dandan Chen, Yufeng Xue, Wanqiu Wang, Wen Liu, Wei-Hua Tang, and Zhijun Tang
- Subjects
chemistry.chemical_classification ,Amidohydrolase ,food and beverages ,General Chemistry ,Lyase ,Biochemistry ,Catalysis ,Virulence factor ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Enzyme ,Biosynthesis ,chemistry ,Host adaptation ,Gene ,Bond cleavage - Abstract
Fusarium graminearum is a pathogenic fungus causing huge economic losses worldwide via crop infection leading to yield reduction and grain contamination. The process through which the fungal invasion occurs remains poorly understood. We recently characterized fusaoctaxin A in F. graminearum, where this octapeptide virulence factor results from an assembly line encoded in fg3_54, a gene cluster proved to be involved in fungal pathogenicity and host adaptation. Focusing on genes in this cluster that are related to fungal invasiveness but not to the biosynthesis of fusaoctaxin A, we here report the identification and characterization of fusaoctaxin B, a new octapeptide virulence factor with comparable activity in wheat infection. Fusaoctaxin B differs from fusaoctaxin A at the N-terminus by possessing a guanidinoacetic acid (GAA) unit, formation of which depends on the combined activities of the protein products of fgm1-3. Fgm1 is a cytochrome P450 protein that oxygenates l-Arg to 4(R)-hydroxyl-l-Arg in a regio- and stereoselective manner. Then, Cβ-Cγ bond cleavage proceeds in the presence of Fgm3, a pyridoxal-5'-phosphate-dependent lyase, giving guanidinoacetaldehyde and l-Ala. Rather than being directly oxidized to GAA, the guanidine-containing aldehyde undergoes spontaneous cyclization and subsequent enzymatic dehydrogenation to provide glycociamidine, which is linearized by Fgm2, a metallo-dependent amidohydrolase. The GAA path in F. graminearum is distinct from that previously known to involve l-Arg:l-Gly aminidotransferase activity. To provide this nonproteinogenic starter unit that primes nonribosomal octapeptidyl assembly, F. graminearum employs new chemistry to process l-Arg through inert C-H bond activation, selective C-C bond cleavage, cyclization-based alcohol dehydrogenation, and amidohydrolysis-associated linearization.
- Published
- 2021
25. Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1
- Author
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Ilia G. Denisov, Stephen G. Sligar, Michael C. Gregory, Yilin Liu, Yelena V. Grinkova, and James R. Kincaid
- Subjects
Stereochemistry ,Hydroxylation ,Spectrum Analysis, Raman ,Polymorphism, Single Nucleotide ,Biochemistry ,Translocation, Genetic ,Article ,Substrate Specificity ,chemistry.chemical_compound ,Catalytic Domain ,medicine ,Humans ,Progesterone ,Bond cleavage ,biology ,Androstenedione ,Steroid 17-alpha-Hydroxylase ,Substrate (chemistry) ,Active site ,Cytochrome P450 ,Hydrogen Bonding ,Dehydroepiandrosterone ,Lyase ,chemistry ,CYP17A1 ,Pregnenolone ,Mutation ,Androgens ,biology.protein ,Steroids ,medicine.drug - Abstract
Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon–carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C(17)–C(20) bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon–carbon bond scission.
- Published
- 2021
26. Simulated gastrointestinal digestion of protein alginate complexes:effects of whey protein cross-linking and the composition and degradation of alginate
- Abstract
Alginate and whey protein are common additives in food production improving storage stability, texture and nutritional value. Alginate forms complexes with whey protein and inhibits proteolysis by pepsin and trypsin, but the influence of alginate protein complexation on digestion is poorly understood. This study shows that whey protein cross-linking by microbial transglutaminase dramatically decreased particle size (2-fold) and viscosity of alginate protein complexes. The INFOGEST in vitro simulated gastrointestinal digestion of whey protein was increased by cross-linking (16%) and suppressed by alginate, most pronounced with high mannuronic acid and least with high guluronic acid content. Sizes of alginate whey protein particles increased during gastric digestion, whereas for cross-linked whey protein complexes the size initially increased, but returned to their initial size at the end of gastric digestion. While alginate is not degraded by human enzymes, a few gut bacteria were recently found to encode lyases and other enzymes metabolizing alginate. Alginate lyase added to the intestinal phase enhanced digestion (9%) as controlled by alginate composition and enzyme specificity. Thus we provide evidence that use of hydrocolloids and processing of protein strongly influence digestion and should be considered when using food additives.
- Published
- 2022
27. Identification of the Catalytic Residues in the Cyclase Domain of the Class IV Lanthipeptide Synthetase SgbL
- Author
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Roderich D. Süssmuth and Julian D. Hegemann
- Subjects
chemistry.chemical_classification ,Stereochemistry ,Organic Chemistry ,Lyase ,Biochemistry ,Cyclase ,Substrate Specificity ,chemistry.chemical_compound ,Enzyme ,Protein Domains ,chemistry ,Biosynthesis ,Dehydroalanine ,Biocatalysis ,Phosphoserine ,Molecular Medicine ,Phosphothreonine ,Peptide Synthases ,Molecular Biology ,Adenylyl Cyclases - Abstract
Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are subdivided into different classes based on their processing enzymes. The three-domain class IV lanthipeptide synthetases (LanL enzymes) consist of N-terminal lyase, central kinase, and C-terminal cyclase domains. While the catalytic residues of the kinase domains (mediating ATP-dependent Ser/Thr phosphorylations) and the lyase domains (carrying out subsequent phosphoserine/phosphothreonine (pSer/pThr) eliminations to yield dehydroalanine/dehydrobutyrine (Dha/Dhb) residues) have been characterized previously, such studies are missing for LanL cyclase domains. To close this gap of knowledge, this study reports on the identification and validation of the catalytic residues in the cyclase domain of the class IV lanthipeptide synthetase SgbL, which facilitate the nucleophilic attacks by Cys thiols on Dha/Dhb residues for the formation of β-thioether crosslinks.
- Published
- 2021
28. Forrestiacids A and B, Pentaterpene Inhibitors of ACL and Lipogenesis: Extending the Limits of Computational NMR Methods in the Structure Assignment of Complex Natural Products
- Author
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Mark T. Hamann, Pankaj Pandey, Amar G. Chittiboyina, Yeun-Mun Choo, Yi Zang, Ting Huang, Ze-Yu Zhao, Jin Feng Hu, Yu-Hang He, Xiaojuan Wang, Hao-Wen Jiang, Pengjun Zhou, Juan Xiong, and Jia Li
- Subjects
Biological Products ,Circular dichroism ,Magnetic Resonance Spectroscopy ,Bicyclic molecule ,Terpenes ,Stereochemistry ,Lipogenesis ,Molecular Conformation ,General Chemistry ,Lyase ,Lanostane ,Catalysis ,Adduct ,chemistry.chemical_compound ,chemistry ,ATP Citrate (pro-S)-Lyase ,Humans ,Moiety ,Molecule ,Enzyme Inhibitors ,Abietane - Abstract
Forrestiacids A (1) and B (2) are a novel class of [4+2] type pentaterpenoids derived from a rearranged lanostane moiety (dienophile) and an abietane unit (diene). These unprecedented molecules were isolated using guidance by molecular ion networking (MoIN) from Pseudotsuga forrestii, an endangered member of the Asian Douglas Fir Family. The intermolecular hetero-Diels-Alder adducts feature an unusual bicyclo[2.2.2]octene ring system. Their structures were elucidated by spectroscopic analysis, GIAO NMR calculations and DP4+ probability analyses, electronic circular dichroism calculations, and X-ray diffraction analysis. This unique addition to the pentaterpene family represents the largest and the most complex molecule successfully assigned using computational approaches to predict accurately chemical shift values. Compounds 1 and 2 exhibited potent inhibitory activities (IC50 s
- Published
- 2021
29. Enzymatic production of N-acetylneuraminic acid: advances and perspectives
- Author
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Xueqin Lv, Guocheng Du, Samra Basharat, Long Liu, Muhammad Iftikhar Hussain, Umar Shahbaz, Zhang Xiaolong, Jianghua Li, and Yanfeng Liu
- Subjects
chemistry.chemical_classification ,chemistry.chemical_compound ,Enzyme ,Biosynthesis ,chemistry ,Biochemistry ,Yield (chemistry) ,food and beverages ,Protein engineering ,Lyase ,Chemical synthesis ,N-Acetylneuraminic acid ,Sialic acid - Abstract
N-Acetyl-d-neuraminic acid (NeuAc), a well-known and well-studied sialic acid, is found in cell surface glycolipids and glycoproteins, where it performs a variety of biological functions. The use of NeuAc as a nutraceutical for infant brain development and as an intermediate for pharmaceutical production demands its production on an industrial scale. Natural extraction, chemical synthesis, enzymatic synthesis, and biosynthesis are the methods used for NeuAc production. Among these methods, enzymatic synthesis using N-acetyl-glucosamine (GlcNAc) 2-epimerase (AGE) for epimerization and N-acetyl-d-neuraminic acid lyase (NAL) for aldol condensation, has been reported to produce NeuAc with high production efficiency. In this review, we discuss advances in the two-step enzymatic synthesis of NeuAc using pyruvate and GlcNAc as substrates. The major challenges in producing NeuAc with high yield are highlighted, including multiple parameter-dependent processes, undesirable reversibility, and diminished solubility of AGEs and NALs. Further, different strategies applied to overcome the limitations of the two-step enzymatic production are discussed, such as pyruvate concentration and temperature shift during the process to increase conversion yield, use of mathematical and computational simulations for process optimization, enzyme engineering to make enzymes highly efficient, and the use of tags and chaperones to increase enzyme solubility. We suggest future directions and the strategies that can be followed to improve enzymatic synthesis of NeuAc.
- Published
- 2021
30. Protein Domain Specific Covalent Inhibition of Human DNA Polymerase β
- Author
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Marc M. Greenberg, Shelby C Yuhas, and Ananya Majumdar
- Subjects
DNA damage ,DNA repair ,DNA polymerase ,Mutant ,Biochemistry ,DNA polymerases ,chemistry.chemical_compound ,Very Important Paper ,enzyme inhibition ,Molecular Biology ,DNA Polymerase beta ,Polymerase ,biology ,Communication ,Organic Chemistry ,Base excision repair ,Lyase ,Communications ,chemistry ,biology.protein ,covalent inhibitors ,Molecular Medicine ,DNA - Abstract
DNA polymerase β (Pol β) is a frequently overexpressed and/or mutated bifunctional repair enzyme. Pol β possesses polymerase and lyase active sites, that are employed in two steps of base excision repair. Pol β is an attractive therapeutic target for which there is a need for inhibitors. Two mechanistically inspired covalent inhibitors (1, IC50=21.0 μM; 9, IC50=18.7 μM) that modify lysine residues in different Pol β active sites are characterized. Despite modifying lysine residues in different active sites, 1 and 9 inactivate the polymerase and lyase activities of Pol β. Fluorescence anisotropy experiments indicate that they do so by preventing DNA binding. Inhibitors 1 and 9 provide the basis for a general approach to preparing domain selective inhibitors of bifunctional polymerases. Such molecules could prove to be useful tools for studying the role of wild type and mutant forms of Pol β and other polymerases in DNA repair., A single synthesis and screening strategy yields covalent inhibitors that inactivate bifunctional DNA polymerase β by modifying lysine residues in different active site domains and prevent DNA binding.
- Published
- 2021
31. The Promiscuous Activity of the Radical <scp>SAM</scp> Enzyme <scp>NosL</scp> toward Two Unnatural Substrates
- Author
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Qi Zhang, Xinjian Ji, and Yue Yin
- Subjects
chemistry.chemical_classification ,chemistry.chemical_compound ,Enzyme ,Biosynthesis ,chemistry ,Biochemistry ,General Chemistry ,Lyase ,Radical SAM ,Enzyme catalysis - Published
- 2021
32. Conjugates of methionine γ-lyase with polysialic acid: Two approaches to antitumor therapy
- Author
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Elena A. Morozova, Tatyana V. Demidkina, Natalya V. Anufrieva, V. Timofeeva, A. Solovieva, Vasily Koval, E. Lesnova, S.V. Revtovich, A. Kushch, and Vitalia V. Kulikova
- Subjects
Antineoplastic Agents ,02 engineering and technology ,Conjugated system ,Biochemistry ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Cell Line, Tumor ,Neoplasms ,Animals ,Humans ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,Mice, Inbred BALB C ,0303 health sciences ,Methionine ,Polysialic acid ,General Medicine ,Metabolism ,021001 nanoscience & nanotechnology ,Lyase ,In vitro ,Carbon-Sulfur Lyases ,Kinetics ,Enzyme ,chemistry ,Cancer cell ,MCF-7 Cells ,Sialic Acids ,Female ,0210 nano-technology - Abstract
The methionine dependence is a well known phenomenon in metabolism of cancer cells. Methionine γ-lyase (EC 4.4.1.11, MGL) catalyzes the γ-elimination reaction of L-methionine and thus could effectively inhibit the growth of malignant cells. Recently we have demonstrated that the mutant form of the enzyme C115H MGL can be used as a component of the pharmacological pair enzyme/S-(allyl/alkyl)-L-cysteine sulfoxides to yield thiosulfinates in situ. Thiosulfinates were shown to be toxic to various cancer cell lines. Therefore the application of the enzyme in enzyme pro-drug therapy may be promising. The conjugates of MGL and C115H MGL with polysialic acid were obtained and their kinetic and pharmacokinetic parameters were determined. The formation of polysialic shell around the enzyme was confirmed by atomic force microscopy. The half-life of conjugated enzymes increased 3–6 times compared to the native enzyme. The cytotoxic effect of conjugated MGL against methionine dependent cancer cell lines was increased two times compared to the values for the native enzymes. The anticancer efficiency of thiosulfinates produced by pharmacological pair C115H MGL/S-(allyl/alkyl)-L-cysteine sulfoxides was demonstrated in vitro. The results indicate that the conjugates of MGL with polysialic acid could be new antitumor drugs.
- Published
- 2021
33. Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and β-d-GlcA
- Author
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Ryoji Usami, Ichiro Suzuki, Yuta Kawasaki, Minoru Takeda, Izuru Kawamura, Masato Katahira, Tadashi Nittami, Daisuke Kashiwabara, Daisuke Kan, Keiko Kondo, and Michio Sato
- Subjects
Disulfide Linkage ,Glycoconjugate ,Stereochemistry ,Peptide ,Sphaerotilus ,02 engineering and technology ,Polysaccharide ,Biochemistry ,03 medical and health sciences ,Polysaccharides ,Structural Biology ,Molecular Biology ,Sphaerotilus montanus ,Polysaccharide-Lyases ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,integumentary system ,biology ,Sphaerotilus natans ,General Medicine ,Sheath ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Lyase ,Enzyme ,chemistry ,Thiopeptidoglycan lyase ,0210 nano-technology ,Paenibacillus ,Cysteine - Abstract
Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-β- d -GlcA-(1→4)-β- d -Glc-(1→3)-β- d -GalNAc-(1→4)-α- d -GalNAc-(1→4)-α- d -GalN-(1→]n (β- d -Glc and α- d -GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α- d -GalN and β- d -GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.
- Published
- 2021
34. Structures of topoisomerase V in complex with DNA reveal unusual DNA-binding mode and novel relaxation mechanism
- Author
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Alfonso Mondragón and A. Osterman
- Subjects
Conformational change ,biology ,General Immunology and Microbiology ,DNA repair ,Chemistry ,Topoisomerase ,General Neuroscience ,Active site ,Processivity ,DNA ,General Medicine ,Euryarchaeota ,Lyase ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,DNA Topoisomerases, Type I ,biology.protein ,Biophysics ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,A-DNA - Abstract
Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.
- Published
- 2022
35. Biochemical characterization of an ulvan lyase from the marine flavobacterium Formosa agariphila KMM 3901T.
- Author
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Reisky, Lukas, Stanetty, Christian, Mihovilovic, Marko D., Schweder, Thomas, Hehemann, Jan-Hendrik, and Bornscheuer, Uwe T.
- Subjects
- *
LYASES , *FLAVOBACTERIUM , *BIOCHEMISTRY , *CARBON dioxide , *ALGAE biodegradation , *POLYSACCHARIDES , *DEPOLYMERIZATION , *MARINE bacteria - Abstract
Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast array of carbohydrate-active enzymes. These enzymes are important tools to establish biotechnological processes based on algal biomass. Green tides, which cover coastal areas with huge amounts of algae from the genus Ulva, represent a globally rising problem, but also an opportunity because their biomass could be used in biorefinery processes. One major component of their cell walls is the anionic polysaccharide ulvan for which the enzymatic depolymerization remains largely unknown. Ulvan lyases catalyze the initial depolymerization step of this polysaccharide, but only a few of these enzymes have been described. Here, we report the cloning, overexpression, purification, and detailed biochemical characterization of the endolytic ulvan lyase from Formosa agariphila KMM 3901T which is a member of the polysaccharide lyase family PL28. The identified biochemical parameters of the ulvan lyase reflect adaptation to the temperate ocean where the bacterium was isolated from a macroalgal surface. The NaCl concentration has a high influence on the turnover number of the enzyme and the affinity to ulvan. Divalent cations were shown to be essential for enzyme activity with Ca2+ likely being the native cofactor of the ulvan lyase. This study contributes to the understanding of ulvan lyases, which will be useful for future biorefinery applications of the abundant marine polysaccharide ulvan. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
36. Biochemical characterization of an ulvan lyase from the marine flavobacterium Formosa agariphila KMM 3901T.
- Author
-
Reisky, Lukas, Stanetty, Christian, Mihovilovic, Marko D., Schweder, Thomas, Hehemann, Jan-Hendrik, and Bornscheuer, Uwe T.
- Subjects
LYASES ,FLAVOBACTERIUM ,BIOCHEMISTRY ,CARBON dioxide ,ALGAE biodegradation ,POLYSACCHARIDES ,DEPOLYMERIZATION ,MARINE bacteria - Abstract
Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast array of carbohydrate-active enzymes. These enzymes are important tools to establish biotechnological processes based on algal biomass. Green tides, which cover coastal areas with huge amounts of algae from the genus Ulva, represent a globally rising problem, but also an opportunity because their biomass could be used in biorefinery processes. One major component of their cell walls is the anionic polysaccharide ulvan for which the enzymatic depolymerization remains largely unknown. Ulvan lyases catalyze the initial depolymerization step of this polysaccharide, but only a few of these enzymes have been described. Here, we report the cloning, overexpression, purification, and detailed biochemical characterization of the endolytic ulvan lyase from Formosa agariphila KMM 3901
T which is a member of the polysaccharide lyase family PL28. The identified biochemical parameters of the ulvan lyase reflect adaptation to the temperate ocean where the bacterium was isolated from a macroalgal surface. The NaCl concentration has a high influence on the turnover number of the enzyme and the affinity to ulvan. Divalent cations were shown to be essential for enzyme activity with Ca2+ likely being the native cofactor of the ulvan lyase. This study contributes to the understanding of ulvan lyases, which will be useful for future biorefinery applications of the abundant marine polysaccharide ulvan. [ABSTRACT FROM AUTHOR]- Published
- 2018
- Full Text
- View/download PDF
37. Identification of a unique insertion in plant organellar DNA polymerases responsible for 5′-dRP lyase and strand-displacement activities: Implications for Base Excision Repair.
- Author
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Trasviña-Arenas, Carlos H., Baruch-Torres, Noe, Cordoba-Andrade, Francisco J., Ayala-García, Víctor M., García-Medel, Paola L., Díaz-Quezada, Corina, Peralta-Castro, Antolín, Ordaz-Ortiz, José Juan, and Brieba, Luis G.
- Subjects
- *
PLANT mitochondria , *CHLOROPLAST DNA , *DNA polymerases , *ARABIDOPSIS thaliana , *POLYMERIZATION - Abstract
Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5′-deoxyribose phosphate (5′-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5′-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5′-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5′-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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- View/download PDF
38. Crystal structure of the nitrosuccinate lyase CreD in complex with fumarate provides insights into the catalytic mechanism for nitrous acid elimination.
- Author
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Katsuyama, Yohei, Sato, Yukari, Sugai, Yoshinori, Higashiyama, Yousuke, Senda, Miki, Senda, Toshiya, and Ohnishi, Yasuo
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- *
SUCCINATES , *LYASES , *BISOPROLOL , *NITROUS acid , *CRYSTAL structure - Abstract
Enzymes belonging to the aspartase/fumarase superfamily catalyze elimination of various functional groups from succinate derivatives and play an important role in primary metabolism and aromatic compound degradation. Recently, an aspartase/fumarase superfamily enzyme, CreD, was discovered in cremeomycin biosynthesis. This enzyme catalyzes the elimination of nitrous acid from nitrosuccinate synthesized from aspartate by CreE, a flavin‐dependent monooxygenase. Nitrous acid generated by this pathway is an important precursor of the diazo group of cremeomycin. CreD is the first aspartase/fumarase superfamily enzyme that was reported to catalyze the elimination of nitrous acid, and therefore we aimed to analyze its reaction mechanism. The crystal structure of CreD was determined by the molecular replacement native‐single anomalous diffraction method at 2.18 Å resolution. Subsequently, the CreD‐fumarate complex structure was determined at 2.30 Å resolution by the soaking method. Similar to other aspartase/fumarase superfamily enzymes, the crystal structure of CreD was composed of three domains and formed a tetramer. Two molecules of fumarate were observed in one subunit of the CreD‐fumarate complex. One of them was located in the active site pocket formed by three different subunits. Intriguingly, no histidine residue, which usually functions as a catalytic acid in aspartase/fumarase superfamily enzymes, was found around the fumarate molecule in the active site. Based on the mutational analysis, we propose a catalytic mechanism of CreD, in which Arg325 acts as a catalytic acid. Databases: The crystal structures of CreD and the CreD‐fumarate complex were deposited to PDB under the accession numbers 5XNY and 5XNZ, respectively. Enzymes: Nitrosuccinate lyase CreD, EC4.3 [ABSTRACT FROM AUTHOR]
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- 2018
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39. Green synthesis of magnetite nanoparticle and its regulatory effect on fermentative hydrogen production from lignocellulosic hydrolysate by Klebsiella sp
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Yanbin Li, Juanjuan Cao, Qin Zhang, Siyuan Xu, Pengfei Ding, and Yonggui Zhang
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Hydrogenase ,Renewable Energy, Sustainability and the Environment ,Chemistry ,Energy Engineering and Power Technology ,Nanoparticle ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Lyase ,01 natural sciences ,Combinatorial chemistry ,Hydrolysate ,0104 chemical sciences ,Metabolic pathway ,chemistry.chemical_compound ,Fuel Technology ,Fermentative hydrogen production ,0210 nano-technology ,Hydrogen production ,Magnetite - Abstract
This study describes the synthesis and characterization of magnetite nanoparticles (NPs) from water hyacinth (WH) extract and its regulatory effect on fermentative hydrogen production from lignocellulosic hydrolysate by Klebsiella sp. Characterization of WH-magnetite-NP revealed that it was a pure magnetite NP in a spherical shape with an average particle size of 13.5 ± 3.7 nm. The maximum cumulative hydrogen production with an increment of 23.49% and an optimum Y(H2/S) of 83.20 ± 2.19 mL/gsubstrate was obtained with WH-magnetite-NP at 20 mg/L. Monitoring of key node metabolites further established the potential of WH-magnetite-NP to increase the flux distribution of the hydrogen synthesis pathway. The hydrogenase activity was enhanced via WH-magnetite-NP addition, with peak value 2.1 times of the control. The expression of functional genes in key pathways assessed via RT-PCR highlighted the effect of WH-magnetite-NP on the evident promotion of hydrogenase and formate-hydrogen lyase. This is the first attempt to detect the expression of multiple functional genes in key metabolic pathways to explain the regulatory mechanism upon NP addition.
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- 2021
40. Production of octanoic acid in Saccharomyces cerevisiae : Investigation of new precursor supply engineering strategies and intrinsic limitations
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Florian Wernig, Leonie Baumann, Eckhard Boles, and Mislav Oreb
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0106 biological sciences ,0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Phosphoketolase ,Bioengineering ,Pentose phosphate pathway ,01 natural sciences ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,chemistry.chemical_compound ,ddc:570 ,010608 biotechnology ,chemistry.chemical_classification ,biology ,Acetyl-CoA ,Fatty acid ,Lyase ,biology.organism_classification ,Fatty acid synthase ,030104 developmental biology ,Metabolic Engineering ,Biochemistry ,chemistry ,biology.protein ,Caprylates ,Flux (metabolism) ,Biotechnology - Abstract
The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.
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- 2021
41. Architecture of the membrane-bound cytochrome c heme lyase CcmF
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Lorena Ilcu, Oliver Einsle, Anton Brausemann, and Lin Zhang
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0303 health sciences ,Cofactor binding ,biology ,Cytochrome ,Chemistry ,Stereochemistry ,Thermus thermophilus ,Cytochrome c ,Cell Membrane ,030302 biochemistry & molecular biology ,Lyases ,Cell Biology ,Lyase ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,Heme B ,biology.protein ,Molecular Biology ,Heme ,Integral membrane protein ,030304 developmental biology - Abstract
The covalent attachment of one or multiple heme cofactors to cytochrome c protein chains enables cytochrome c proteins to be used in electron transfer and redox catalysis in extracytoplasmic environments. A dedicated heme maturation machinery, whose core component is a heme lyase, scans nascent peptides after Sec-dependent translocation for CXnCH-binding motifs. Here we report the three-dimensional (3D) structure of the heme lyase CcmF, a 643-amino acid integral membrane protein, from Thermus thermophilus. CcmF contains a heme b cofactor at the bottom of a large cavity that opens toward the extracellular side to receive heme groups from the heme chaperone CcmE for cytochrome maturation. A surface groove on CcmF may guide the extended apoprotein to heme attachment at or near a loop containing the functionally essential WXWD motif, which is situated above the putative cofactor binding pocket. The structure suggests heme delivery from within the membrane, redefining the role of the chaperone CcmE.
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- 2021
42. Structure and Mechanism of <scp>d</scp>-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5′-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a <scp>d</scp>-Amino Acid
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Robert S. Phillips, Samuel C.-K. Ting, and Kaitlin L. Anderson
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Models, Molecular ,Ammonia-Lyases ,Stereochemistry ,Dimer ,Glucose-6-Phosphate ,Lyases ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Article ,Phosphates ,Substrate Specificity ,chemistry.chemical_compound ,Elimination reaction ,Tetramer ,Amino Acids ,Binding site ,Pyridoxal ,Schiff Bases ,Transaminases ,Glucosamine ,Binding Sites ,Substrate (chemistry) ,Lyase ,Kinetics ,chemistry ,Pyridoxal Phosphate ,Dehydratase - Abstract
D-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of D-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D(2)O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.
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- 2021
43. Development of a versatile and efficient C–N lyase platform for asymmetric hydroamination via computational enzyme redesign
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Yifan Bu, Bian Wu, Tong Zhu, Yinghui Wang, Wenya Tian, Xuexian Cui, Tao Li, Ruifeng Li, and Yinglu Cui
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Ammonia-Lyases ,Chemistry ,Process Chemistry and Technology ,Enantioselective synthesis ,Regioselectivity ,Bioengineering ,Lyase ,Biochemistry ,Combinatorial chemistry ,Catalysis ,Nucleophile ,Biocatalysis ,Electrophile ,Hydroamination - Abstract
Although C–N bonds are ubiquitous in natural products, pharmaceuticals and agrochemicals, biocatalysts forging these bonds with high atom-efficiency and enantioselectivity have been limited to a few select enzymes. In particular, ammonia lyases have emerged as powerful catalysts to access C–N bond formation via hydroamination. However, the use of ammonia lyases is rather restricted due to their narrow synthetic scope. Herein, we report the computational redesign of aspartase, a highly specific ammonia lyase, to yield C–N lyases with cross-compatibility of non-native nucleophiles and electrophiles. A wide range of non-canonical amino acids (ncAAs) are afforded with excellent conversion (up to 99%), regioselectivity >99% and enantioselectivity >99%. The process is scalable under industrially relevant protocols (exemplified in kilogram-scale synthesis) and can be facilely integrated in cascade reactions (demonstrated in the synthesis of β-lactams with N-1 and C-4 substitutions). This versatile and efficient C–N lyase platform supports the preparation of ncAAs and their derivatives, and will present opportunities in synthetic biology. Ammonia lyases are powerful catalysts to access C–N bond formation via hydroamination, but show a narrow synthetic scope. Now, by computational redesign of an aspartase, a C–N lyase is developed that shows cross-compatibility of non-native nucleophiles and electrophiles expanding the synthetic scope.
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- 2021
44. High CO2 levels drive the TCA cycle backwards towards autotrophy
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Eugenio Pettinato, Wolfgang Eisenreich, Thomas Steiner, Achim Mall, Ivan A. Berg, Lydia Steffens, and Simone König
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Reverse Krebs cycle ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,Pyruvate synthase ,biology ,030306 microbiology ,Chemistry ,Dehydrogenase ,Tricarboxylic acid ,Lyase ,Citric acid cycle ,03 medical and health sciences ,Metabolic pathway ,Biochemistry ,biology.protein ,Citrate synthase ,030304 developmental biology - Abstract
It has recently been shown that in anaerobic microorganisms the tricarboxylic acid (TCA) cycle, including the seemingly irreversible citrate synthase reaction, can be reversed and used for autotrophic fixation of carbon1,2. This reversed oxidative TCA cycle requires ferredoxin-dependent 2-oxoglutarate synthase instead of the NAD-dependent dehydrogenase as well as extremely high levels of citrate synthase (more than 7% of the proteins in the cell). In this pathway, citrate synthase replaces ATP-citrate lyase of the reductive TCA cycle, which leads to the spending of one ATP-equivalent less per one turn of the cycle. Here we show, using the thermophilic sulfur-reducing deltaproteobacterium Hippea maritima, that this route is driven by high partial pressures of CO2. These high partial pressures are especially important for the removal of the product acetyl coenzyme A (acetyl-CoA) through reductive carboxylation to pyruvate, which is catalysed by pyruvate synthase. The reversed oxidative TCA cycle may have been functioning in autotrophic CO2 fixation in a primordial atmosphere that is assumed to have been rich in CO2. In the deltaproteobacterium Hippea maritima, the tricarboxylic acid (TCA) cycle can be reversed by high partial pressures of CO2 for the autotrophic fixation of carbon.
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- 2021
45. Synthesis of Bempedoic Acid through Electrochemical Decarboxylation of Dialkylated Malonic Acid
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Lifang Tian, Yahui Wang, Yue Zheng, and Zhimin Xu
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010405 organic chemistry ,Chemistry ,Decarboxylation ,Organic Chemistry ,Ketone formation ,Malonic acid ,010402 general chemistry ,Electrochemistry ,Lyase ,01 natural sciences ,Adenosine ,Catalysis ,0104 chemical sciences ,chemistry.chemical_compound ,Yield (chemistry) ,medicine ,Organic chemistry ,Bempedoic acid ,medicine.drug - Abstract
Bempedoic acid is a small-molecule inhibitor of adenosine triphosphate-citrate lyase (ACL) that is effective in the treatment of hypercholesterolemia and hypertension. In this paper, a new, six-step synthesis of bempedoic acid with 42% overall yield is reported. Ketone formation by electrochemical decarboxylation of dialkylated malonic acid is introduced as the key step of this process. This method uses mild conditions and its high efficiency makes it potentially suitable for industrial production.
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- 2021
46. Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase
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Marco Malatesta, Domenico Acquotti, Alessio Peracchi, Toda Stankovic, Katharina Pallitsch, and Erika Zangelmi
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chemistry.chemical_classification ,Catabolism ,Microorganism ,Phosphorus ,chemistry.chemical_element ,Lyase ,Biochemistry ,Phosphonate degradation ,Ammonia ,chemistry.chemical_compound ,Enzyme ,Marine bacteriophage ,chemistry - Abstract
Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade ...
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- 2021
47. The role of <scp> Escherichia coli FhlA </scp> transcriptional activator in generation of proton motive force and <scp> F O F 1 ‐ATPase </scp> activity at <scp>pH</scp> 7.5
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Anait Vassilian, Satenik Khalatyan, Heghine Gevorgyan, and Karen Trchounian
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Glycerol ,0301 basic medicine ,Proton ATPase ,Formates ,Clinical Biochemistry ,Mutant ,Acetates ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Escherichia coli ,Genetics ,medicine ,Formate ,Molecular Biology ,Chemiosmosis ,Escherichia coli Proteins ,Proton-Motive Force ,Cell Biology ,Hydrogen-Ion Concentration ,Lyase ,Carbon ,Proton-Translocating ATPases ,Glucose ,030104 developmental biology ,Dicyclohexylcarbodiimide ,chemistry ,030220 oncology & carcinogenesis ,Fermentation ,Trans-Activators ,Oxidation-Reduction ,Hydrogen - Abstract
Escherichia coli is able to utilize the mixture of carbon sources and produce molecular hydrogen (H2 ) via formate hydrogen lyase (FHL) complexes. In current work role of transcriptional activator of formate regulon FhlA in generation of fermentation end products and proton motive force, N'N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity at 20 and 72 hr growth during utilization of mixture of glucose, glycerol, and formate were investigated. It was shown that in fhlA mutant specific growth rate was ~1.5 fold lower compared to wt, while addition of DCCD abolished the growth in fhlA but not in wt. Formate was not utilized in fhlA mutant but wt cells simultaneously utilized formate with glucose. Glycerol utilization started earlier (from 2 hr) in fhlA than in wt. The DCCD-sensitive ATPase activity in wt cells membrane vesicles increased ~2 fold at 72 hr and was decreased 70% in fhlA. Addition of formate in the assays increased proton ATPase activity in wt and mutant strain. FhlA absence mainly affected the ΔpH but not ΔΨ component of Δp in the cells grown at 72 hr but not in 24 hr. The Δp in wt cells decreased from 24 to 72 hr of growth ~40 mV while in fhlA mutant it was stable. Taken together, it is suggested that FhlA regulates the concentration of fermentation end products and via influencing FO F1 -ATPase activity contributes to the proton motive force generation.
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- 2021
48. Hydrogen sulphide reduces hyperhomocysteinaemia‐induced endothelial ER stress by sulfhydrating protein disulphide isomerase to attenuate atherosclerosis
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Shan Jiang, Xiaofang Fan, Zhenzhen Chen, Wenjing Xu, Yongsheng Gong, Changting Cui, Jun Cai, and Bin Geng
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Male ,0301 basic medicine ,Apolipoprotein E ,Homocysteine ,Mice, Knockout, ApoE ,Hyperhomocysteinemia ,Protein Disulfide-Isomerases ,Cell Line ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,protein disulphide isomerase ,medicine ,Animals ,Humans ,Hydrogen Sulfide ,Endothelial dysfunction ,Chemistry ,Endoplasmic reticulum ,Endothelial Cells ,Original Articles ,homocysteine ,Cell Biology ,equipment and supplies ,Atherosclerosis ,Endoplasmic Reticulum Stress ,medicine.disease ,Lyase ,Up-Regulation ,Cell biology ,Disease Models, Animal ,HEK293 Cells ,030104 developmental biology ,030220 oncology & carcinogenesis ,Knockout mouse ,Unfolded protein response ,sulfhydration ,Molecular Medicine ,Original Article ,hydrogen sulphide ,Cysteine - Abstract
Hyperhomocysteinaemia (HHcy)‐impaired endothelial dysfunction including endoplasmic reticulum (ER) stress plays a crucial role in atherogenesis. Hydrogen sulphide (H2S), a metabolic production of Hcy and gasotransmitter, exhibits preventing cardiovascular damages induced by HHcy by reducing ER stress, but the underlying mechanism is unclear. Here, we made an atherosclerosis with HHcy mice model by ApoE knockout mice and feeding Pagien diet and drinking L‐methionine water. H2S donors NaHS and GYY4137 treatment lowered plaque area and ER stress in this model. Protein disulphide isomerase (PDI), a modulation protein folding key enzyme, was up‐regulated in plaque and reduced by H2S treatment. In cultured human aortic endothelial cells, Hcy dose and time dependently elevated PDI expression, but inhibited its activity, and which were rescued by H2S. H2S and its endogenous generation key enzyme‐cystathionine γ lyase induced a new post‐translational modification‐sulfhydration of PDI. Sulfhydrated PDI enhanced its activity, and two cysteine‐terminal CXXC domain of PDI was identified by site mutation. HHcy lowered PDI sulfhydration association ER stress, and H2S rescued it but this effect was blocked by cysteine site mutation. Conclusively, we demonstrated that H2S sulfhydrated PDI and enhanced its activity, reducing HHcy‐induced endothelial ER stress to attenuate atherosclerosis development.
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- 2021
49. Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b5
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Stephen G. Sligar, James R. Kincaid, Ilia G. Denisov, and Yilin Liu
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biology ,Hydrogen bond ,Stereochemistry ,Chemistry ,Allosteric regulation ,Substrate (chemistry) ,Active site ,General Chemistry ,010402 general chemistry ,Lyase ,01 natural sciences ,Biochemistry ,Catalysis ,0104 chemical sciences ,Colloid and Surface Chemistry ,Cytochrome b5 ,biology.protein ,Lyase activity ,Bond cleavage - Abstract
CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b5 (Mn-Cytb5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-b5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.
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- 2021
50. Isocitrate dehydrogenase 1 from Acinetobacter baummanii (AbIDH1) enzymatic characterization and its regulation by phosphorylation
- Author
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Meng-li Wang, Peng Wang, Ping Song, Guoping Zhu, and Qing-yang Zheng
- Subjects
Acinetobacter baumannii ,inorganic chemicals ,0301 basic medicine ,Phosphatase ,Mutation, Missense ,Biochemistry ,Michaelis–Menten kinetics ,03 medical and health sciences ,Bacterial Proteins ,Enzyme kinetics ,Cloning, Molecular ,Phosphorylation ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Kinase ,General Medicine ,Lyase ,Isocitrate Dehydrogenase ,030104 developmental biology ,Isocitrate dehydrogenase ,Enzyme ,Amino Acid Substitution ,chemistry ,Mutagenesis, Site-Directed ,bacteria - Abstract
Acinetobacter baumannii encodes all enzymes required in the tricarboxylic acid (TCA) cycle and glyoxylate bypass except for isocitrate dehydrogenase kinase/phosphatase (IDHKP), which can phosphorylate isocitrate dehydrogenase (IDH) at a substrate-binding Ser site and control the carbon flux in enterobacteria, such as Escherichia coli. The potential kinase was not successfully pulled down from A. baumannii cell lyase; therefore, whether the IDH 1 from A. baumannii (AbIDH1) can be phosphorylated to regulate intracellular carbon flux has not been clarified. Herein, the AbIDH1 gene was cloned, the encoded protein was expressed and purified to homogeneity, and phosphorylation and enzyme kinetics were evaluated in vitro. Gel filtration and SDS-PAGE analyses showed that AbIDH1 is an 83.5 kDa homodimer in solution. The kinetics showed that AbIDH1 is a fully active NADP-dependent enzyme. The Michaelis constant Km is 46.6 (Mn2+) and 18.1 μM (Mg2+) for NADP+ and 50.5 (Mn2+) and 65.4 μM (Mg2+) for the substrate isocitrate. Phosphorylation experiments in vitro indicated that AbIDH1 is a substrate for E. coli IDHKP. The activity of AbIDH1 treated with E. coli IDHKP immediately decreased by 80% within 9 min. Mass spectrometry indicated that the conserved Ser113 of AbIDH1 is phosphorylated. Continuous phosphorylation-mimicking mutants (Ser113Glu and Ser113Asp) lack almost all enzymatic activity. Side-chain mutations at Ser113 (Ser113Thr, Ser113Ala, Ser113Gly and Ser113Tyr) remarkably reduce the enzymatic activity. Understanding the potential of AbIDH1 phosphorylation enables further investigations of the AbIDH1 physiological functions in A. baumannii.
- Published
- 2021
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