72 results on '"Kazunari Yoneda"'
Search Results
2. Effect of Phytic Acid Dietary Level on Growth Performance and Serum Components in Broiler Chickens
- Author
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Takeshi Shibata, Kazunari Yoneda, Tomohiro Araki, and Takahiro Nikki
- Subjects
abdominal fat ,chicken ,ip6 ,phytic acid ,Animal culture ,SF1-1100 - Abstract
This study was conducted to elucidate the effects of dietary phytic acid supplementation on organs and serum components in broiler chickens. A total of 30 1-day-old broiler chicks were divided into three treatment groups (n=10, each). The control group was fed normal diet and the other groups were fed diet supplemented with 0.06% and 0.12% phytic acid for 30 days. No differences in body and muscle weights and 21 serum biochemical parameters were detected between phytic acid treatments and the control. However, abdominal fat weight decreased significantly with 0.12% phytic acid treatment (P
- Published
- 2012
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3. Stereospecificity of hydride transfer and molecular docking in FMN‐dependent NADH‐indigo reductase of Bacillus smithii
- Author
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Haruhiko Sakuraba, Tomohiro Araki, Kazunari Yoneda, and Toshihisa Ohshima
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0301 basic medicine ,FMN Reductase ,Stereochemistry ,QH301-705.5 ,Flavin Mononucleotide ,Flavin mononucleotide ,Bacillus ,Reductase ,FMN‐dependent NADH‐indigo reductase ,Indigo Carmine ,Molecular Docking Simulation ,General Biochemistry, Genetics and Molecular Biology ,Indigo ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Stereospecificity ,Moiety ,Biology (General) ,Research Articles ,Nicotinamide ,Hydride ,NAD ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,cofactor stereospecificity ,H‐NMR ,Research Article - Abstract
In this study, we investigated the stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase expressed by thermophilic Bacillus smithii. We performed 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations to reveal that the pro‐S hydrogen at the C4 position of the nicotinamide moiety in NADH was specifically transferred to the flavin‐N5 atom of FNM. Altogether, our findings may aid in the improvement of the indigo dyeing (Aizome) process., The stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase from the thermophilic bacterium Bacillus smithii was studied. Both analyses of 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations showed that the pro‐S hydrogen of NADH was specifically transferred to the flavin‐N5 atom of FNM.
- Published
- 2021
4. Structural and biochemical characterization of an extremely thermostable FMN-dependent NADH-indigo reductase from Bacillus smithii
- Author
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Misa Yoshioka, Tomohiro Araki, Toshihisa Ohshima, Kazunari Yoneda, and Haruhiko Sakuraba
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FMN Reductase ,Flavin Mononucleotide ,Bacillus ,02 engineering and technology ,Reductase ,Indigo Carmine ,medicine.disease_cause ,Biochemistry ,Catalysis ,Indigo ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Escherichia coli ,medicine ,NADH, NADPH Oxidoreductases ,Cloning, Molecular ,Molecular Biology ,030304 developmental biology ,Thermostability ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Thermophile ,Active site ,General Medicine ,NAD ,021001 nanoscience & nanotechnology ,Molecular Docking Simulation ,Kinetics ,Enzyme ,Indigo carmine ,Mutagenesis, Site-Directed ,biology.protein ,Oxidoreductases ,0210 nano-technology - Abstract
The FMN-dependent NADH-indigo reductase gene from the thermophilic bacterium Bacillus smithii was overexpressed in Escherichia coli. The expressed enzyme functioned as a highly thermostable indigo reductase that retained complete activity even after incubation at 100 °C for 10 min. Furthermore, B. smithii indigo reductase exhibited high stability over a wider pH range and longer storage periods compared with indigo reductases previously identified from other sources. The enzyme catalyzed the reduction of various azo compounds and indigo carmine. The crystal structures of the wild-type enzyme in complex with FMN/N-cyclohexyl-2-aminoethanesulfonate (CHES) and the Y151F mutant enzyme in complex with FMN were determined by the molecular replacement method and refined at resolutions of 1.97 and 1.95 A, respectively. Then, indigo carmine molecule was modeled into the active site using the molecular docking simulation and the binding mode of indigo carmine was elucidated. In addition, the structure of B. cohnii indigo reductase, which is relatively less stable than B. smithii indigo reductase, was constructed by homology modeling. The factor contributing to the considerably higher thermostability of B. smithii indigo reductase was analyzed by comparing its structure with that of B. cohnii indigo reductase, which revealed that intersubunit aromatic interactions (F105-F172′ and F172-F105′) may be responsible for the high thermostability of B. smithii indigo reductase. Notably, site-directed mutagenesis results showed that F105 plays a major role in the intersubunit aromatic interaction.
- Published
- 2020
5. Unique active site formation in a novel galactose 1‐phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum
- Author
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Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, Junji Hayashi, and Tatsuya Ohshida
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Models, Molecular ,Protein Conformation, alpha-Helical ,Hot Temperature ,Gene Expression ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Structural Biology ,Enzyme Stability ,Moiety ,Cloning, Molecular ,chemistry.chemical_classification ,0303 health sciences ,biology ,Galactosephosphates ,030302 biochemistry & molecular biology ,Recombinant Proteins ,Protein Binding ,Archaeal Proteins ,Protein subunit ,Genetic Vectors ,03 medical and health sciences ,Escherichia coli ,medicine ,Humans ,UTP-Hexose-1-Phosphate Uridylyltransferase ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Molecular Biology ,Gene ,030304 developmental biology ,Binding Sites ,Sequence Homology, Amino Acid ,Active site ,Substrate (chemistry) ,Kinetics ,Protein Subunits ,Enzyme ,chemistry ,Galactose ,Mutation ,Pyrobaculum ,biology.protein ,Protein Conformation, beta-Strand ,Protein Multimerization ,Sequence Alignment - Abstract
A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.
- Published
- 2019
6. Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii
- Author
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Ryushi Kawakami, Tatsuya Ohshida, Junji Hayashi, Kazunari Yoneda, Toshio Furumoto, Toshihisa Ohshima, and Haruhiko Sakuraba
- Subjects
Models, Molecular ,Ornithine ,Structural Biology ,Pyridoxal Phosphate ,Humans ,General Medicine ,Pyrococcus horikoshii ,Crystallography, X-Ray ,Molecular Biology ,Biochemistry ,Archaea ,Transaminases ,Substrate Specificity - Abstract
Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT.
- Published
- 2021
7. A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium, Thermotoga maritima
- Author
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Haruhiko Sakuraba, Toshihisa Ohshima, Kazunari Yoneda, Kohei Koba, Tatsuya Ohshida, Taketo Ohmori, and Junji Hayashi
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Threonine ,0301 basic medicine ,Hot Temperature ,Protein Conformation ,Dehydrogenase ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme Stability ,Escherichia coli ,Thermotoga maritima ,Aspartate kinase ,Phosphofructokinase 2 ,Amino Acid Sequence ,Bifunctional ,Molecular Biology ,Homoserine dehydrogenase ,Aspartic Acid ,Sequence Homology, Amino Acid ,030102 biochemistry & molecular biology ,biology ,Organic Chemistry ,General Medicine ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Hyperthermophile ,Kinetics ,030104 developmental biology ,chemistry ,Aspartokinase Homoserine Dehydrogenase ,Biocatalysis ,Electrophoresis, Polyacrylamide Gel ,Bacteria ,Biotechnology - Abstract
The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. l-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by l-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to l-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).
- Published
- 2018
8. Crystal structure of the NADP+ and tartrate-bound complex of l-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis
- Author
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Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, and Tomohiro Araki
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,biology ,Stereochemistry ,Chemistry ,Substrate (chemistry) ,Active site ,Dehydrogenase ,General Medicine ,Protein engineering ,Substrate analog ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,biology.protein ,Molecular Medicine ,Binding site ,Peptide sequence - Abstract
A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP+/sulfate ion at 1.18 A and the structure in complex with NADP+/L-tartrate (substrate analog) at 1.57 A. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP+ and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering.
- Published
- 2018
9. Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase fromLactobacillus buchneri
- Author
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Yuta Mutaguchi, Toshihisa Ohshima, Junji Hayashi, Taketo Ohmori, Yume Minemura, Haruhiko Sakuraba, Kazunari Yoneda, and Noriko Nakagawa
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Isomerase ,Crystallography, X-Ray ,03 medical and health sciences ,Tetramer ,Structural Biology ,Amino Acid Sequence ,Isoleucine ,Amino-acid racemase ,Amino Acid Isomerases ,Lactobacillus buchneri ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Substrate (chemistry) ,Active site ,biology.organism_classification ,Lactobacillus ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Pyridoxal Phosphate ,biology.protein ,Sequence Alignment - Abstract
Crystal structures ofLactobacillus buchneriisoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5′-phosphate (PLP), in complex withN-(5′-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex withN-(5′-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of theL. buchneriisoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-∊-caprolactam racemase fromAchromobactor obaeand γ-aminobutyrate aminotransferase fromEscherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved inL. buchneriisoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine.
- Published
- 2017
10. Complete Amino Acid Sequence of a Copper/Zinc-Superoxide Dismutase from Ginger Rhizome
- Author
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Yuki Nishiyama, Tomohiro Araki, Tamo Fukamizo, and Kazunari Yoneda
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0301 basic medicine ,Curcuma aromatica ,Bioengineering ,Sequence alignment ,Ginger ,Biochemistry ,Analytical Chemistry ,Superoxide dismutase ,03 medical and health sciences ,Tandem Mass Spectrometry ,Enzyme Stability ,Amino Acid Sequence ,Cloning, Molecular ,Peptide sequence ,chemistry.chemical_classification ,Binding Sites ,Base Sequence ,030102 biochemistry & molecular biology ,biology ,Superoxide Dismutase ,Organic Chemistry ,Temperature ,Nucleic acid sequence ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Rhizome ,Amino acid ,Zinc ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,Sequence Alignment ,Copper - Abstract
Superoxide dismutase (SOD) is an antioxidant enzyme protecting cells from oxidative stress. Ginger (Zingiber officinale) is known for its antioxidant properties, however, there are no data on SODs from ginger rhizomes. In this study, we purified SOD from the rhizome of Z. officinale (Zo-SOD) and determined its complete amino acid sequence using N terminal sequencing, amino acid analysis, and de novo sequencing by tandem mass spectrometry. Zo-SOD consists of 151 amino acids with two signature Cu/Zn-SOD motifs and has high similarity to other plant Cu/Zn-SODs. Multiple sequence alignment showed that Cu/Zn-binding residues and cysteines forming a disulfide bond, which are highly conserved in Cu/Zn-SODs, are also present in Zo-SOD. Phylogenetic analysis revealed that plant Cu/Zn-SODs clustered into distinct chloroplastic, cytoplasmic, and intermediate groups. Among them, only chloroplastic enzymes carried amino acid substitutions in the region functionally important for enzymatic activity, suggesting that chloroplastic SODs may have a function distinct from those of SODs localized in other subcellular compartments. The nucleotide sequence of the Zo-SOD coding region was obtained by reverse-translation, and the gene was synthesized, cloned, and expressed. The recombinant Zo-SOD demonstrated pH stability in the range of 5-10, which is similar to other reported Cu/Zn-SODs, and thermal stability in the range of 10-60 °C, which is higher than that for most plant Cu/Zn-SODs but lower compared to the enzyme from a Z. officinale relative Curcuma aromatica.
- Published
- 2017
11. Unique coenzyme binding mode of hyperthermophilic archaealsn-glycerol-1-phosphate dehydrogenase fromPyrobaculum calidifontis
- Author
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Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, Kaori Yamamoto, and Junji Hayashi
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0301 basic medicine ,chemistry.chemical_classification ,biology ,030106 microbiology ,Dehydrogenase ,Biochemistry ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,chemistry ,Structural Biology ,Oxidoreductase ,Ribose ,biology.protein ,Coenzyme binding ,NADPH binding ,NAD+ kinase ,Molecular Biology - Abstract
A gene encoding an sn-glycerol-1-phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (Vmax ) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 A. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2' of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild-type enzyme with NADPH, which suggests that the biased interactions around the C2'-phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)-dependent dehydrogenases. Proteins 2016; 84:1786-1796. © 2016 Wiley Periodicals, Inc.
- Published
- 2016
12. Catalytic properties and crystal structure of thermostable NAD(P)H-dependent carbonyl reductase from the hyperthermophilic archaeon Aeropyrum pernix K1
- Author
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Tomohiro Araki, Kazunari Yoneda, Yudai Fukuda, Toshihisa Ohshima, and Haruhiko Sakuraba
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Models, Molecular ,0301 basic medicine ,Carbonyl Reductase ,Protein Conformation ,Archaeal Proteins ,Aeropyrum ,Bioengineering ,Crystallography, X-Ray ,Applied Microbiology and Biotechnology ,Biochemistry ,Genes, Archaeal ,Avian Proteins ,03 medical and health sciences ,Species Specificity ,Oxidoreductase ,Catalytic Domain ,Enzyme Stability ,Animals ,Aeropyrum pernix ,Amino Acid Sequence ,Cloning, Molecular ,Thermostability ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,030102 biochemistry & molecular biology ,biology ,biology.organism_classification ,Recombinant Proteins ,Hyperthermophile ,Alcohol Oxidoreductases ,Kinetics ,030104 developmental biology ,Enzyme ,chemistry ,Mutagenesis, Site-Directed ,NAD+ kinase ,Chickens ,Biotechnology - Abstract
A gene encoding NAD(P)H-dependent carbonyl reductase (CR) from the hyperthermophilic archaeon Aeropyrum pernix K1 was overexpressed in Escherichia coli. Its product was effectively purified and characterized. The expressed enzyme was the most thermostable CR found to date; the activity remained at approximately 75% of its activity after incubation for 10min up to 90°C. In addition, A. pernix CR exhibited high stability at a wider range of pH values and longer periods of storage compared with CRs previously identified from other sources. A. pernix CR catalyzed the reduction of various carbonyl compounds including ethyl 4-chloro-3-oxobutanoate and 9,10-phenanthrenequinone, similar to the CR from thyroidectomized (Tx) chicken fatty liver. However, A. pernix CR exhibited significantly higher Km values against several substrates than Tx chicken fatty liver CR. The three-dimensional structure of A. pernix CR was determined using the molecular replacement method at a resolution of 2.09Å, in the presence of NADPH. The overall fold of A. pernix CR showed moderate similarity to that of Tx chicken fatty liver CR; however, A. pernix CR had no active-site lid unlike Tx chicken fatty liver CR. Consequently, the active-site cavity in the A. pernix CR was much more solvent-accessible than that in Tx chicken fatty liver CR. This structural feature may be responsible for the enzyme's lower affinity for several substrates and NADPH. The factors contributing to the much higher thermostability of A. pernix CR were analyzed by comparing its structure with that of Tx chicken fatty liver CR. This comparison showed that extensive formation of the intrasubunit ion pair networks, and the presence of the strong intersubunit interaction, is likely responsible for A. pernix CR thermostability. Site-directed mutagenesis showed that Glu99 plays a major role in the intersubunit interaction. This is the first report regarding the characteristics and three-dimensional structure of hyperthermophilic archaeal CR.
- Published
- 2016
13. Catalytic properties and crystal structure of UDP-galactose 4-epimerase-like l-threonine 3-dehydrogenase from Phytophthora infestans
- Author
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Kazunari Yoneda, Kenji Fukui, Rina Nagano, Tomohiro Araki, Haruhiko Sakuraba, Takuya Mikami, and Toshihisa Ohshima
- Subjects
Models, Molecular ,Threonine ,Phytophthora infestans ,Glycine ,Bioengineering ,Crystallography, X-Ray ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Biochemistry ,Catalysis ,UDPglucose 4-Epimerase ,Catalytic Domain ,medicine ,Molecular replacement ,Enzyme Inhibitors ,KEGG ,Escherichia coli ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,Temperature ,Active site ,Substrate (chemistry) ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Molecular Docking Simulation ,carbohydrates (lipids) ,Alcohol Oxidoreductases ,Enzyme ,biology.protein ,NAD+ kinase ,Biotechnology - Abstract
We report, for the first time, the three-dimensional structure and biochemical properties of a UDP-galactose 4-epimerase-like l-threonine 3-dehydrogenase (GalE-like L-ThrDH) from Phytophthora infestans, a plant disease-causing fungus. We identified GalE-like L-ThrDH using Kyoto Encyclopedia of Genes and Genomes (KEGG) database as a candidate target for the development of a new fungicide. The GalE-like L-ThrDH gene was expressed in Escherichia coli, and its product was purified and characterized. N-Acetylglycine was found to act as a competitive inhibitor of the enzyme (Ki =0.18 mM). The crystal structure of the unique hexameric GalE-like L-ThrDH was determined using the molecular replacement method at a resolution of 2.3 A, in the presence of NAD+ and citrate, an analogue of the substrate. Based on the molecular docking simulation, N-acetylglycine molecule was modeled into the active site and the binding mode and inhibition mechanism of N-acetylglycine were elucidated.
- Published
- 2020
14. Asp48 function in the hydrogen-bonding network involving Asp52 of hen egg-white lysozyme
- Author
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Yuya Kawaguchi, Kazunari Yoneda, Tomohiro Araki, and Takao Torikata
- Subjects
Models, Molecular ,Stereochemistry ,Crystal structure ,Crystallography, X-Ray ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,Residue (chemistry) ,Reaction rate constant ,Catalytic Domain ,Enzyme Stability ,Side chain ,Animals ,Molecular Biology ,Guanidine ,Aspartic Acid ,biology ,Hydrogen bond ,Organic Chemistry ,Active site ,Substrate (chemistry) ,Hydrogen Bonding ,General Medicine ,chemistry ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,Muramidase ,Lysozyme ,Chickens ,Biotechnology - Abstract
To characterize the hydrogen-bonding network in lysozyme, we focused on the residue of Asp48 located at the active site in hen egg-white lysozyme. We constructed a mutant lysozyme (D48A) and analyzed using (GlcNAc)3 and chitin-affinity chromatography. The substrate binding of subsites D–F in D48A and the activity against (GlcNAc)5 were decreased. The parameters of substrate binding and rate constants obtained from computer simulations confirmed these changes. In the crystal structure, (GlcNAc)4 was located at the same position as wildtype. However, the side chains of Arg45 and Thr47 at subsites E–F were moved by the replacement. Further, the loss of the hydrogen bond between Asp48 and Ser50 changed the hydrogen-bonding network, and this resulted in an alteration of the side chain of Asn59. This result suggests that the hydrogen-bonding network plays a crucial in the function of Asp52 and of transglycosylation at subsites E–F.
- Published
- 2015
15. Crystal structure of the NADP
- Author
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Kazunari, Yoneda, Haruhiko, Sakuraba, Tomohiro, Araki, and Toshihisa, Ohshima
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Molecular Docking Simulation ,Alcohol Oxidoreductases ,Hot Temperature ,Archaeal Proteins ,Catalytic Domain ,Enzyme Stability ,Pyrobaculum ,Serine ,Crystallography, X-Ray ,Tartrates ,NADP ,Protein Binding ,Substrate Specificity - Abstract
A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP
- Published
- 2017
16. Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase
- Author
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Masahiro Watanabe, Toshihisa Ohshima, Junji Hayashi, Tomonari Seto, Haruhiko Sakuraba, Hironaga Akita, Tamotsu Hoshino, and Kazunari Yoneda
- Subjects
0301 basic medicine ,Models, Molecular ,030106 microbiology ,Mutant ,Lysine ,Amino Acid Motifs ,Dehydrogenase ,D-amino acid dehydrogenase ,Protein Engineering ,Applied Microbiology and Biotechnology ,Substrate Specificity ,03 medical and health sciences ,Bacterial Proteins ,Oxidoreductase ,Amino Acid Sequence ,Enzymology and Protein Engineering ,chemistry.chemical_classification ,Binding Sites ,Ecology ,Chemistry ,Substrate (chemistry) ,Oxidative deamination ,Kinetics ,030104 developmental biology ,Biochemistry ,Planococcaceae ,Mutagenesis, Site-Directed ,NAD+ kinase ,Amino Acid Oxidoreductases ,NADP ,Food Science ,Biotechnology - Abstract
A stable NADP + -dependent d -amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d -phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d -leucine, d -norleucine, d -methionine, d -isoleucine, and d -tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP + and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d -amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d -amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d -amino acids, but all include tedious steps. The use of NAD(P) + -dependent d -amino acid dehydrogenase (DAADH) makes single-step production of d -amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d -amino acids, such as d -leucine and d -isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d -amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.
- Published
- 2017
17. Structure of<scp>D</scp>-tagatose 3-epimerase-like protein fromMethanocaldococcus jannaschii
- Author
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Haruhiko Sakuraba, Toshihisa Ohshima, Kazunari Yoneda, Goro Takata, and Keiko Uechi
- Subjects
Models, Molecular ,Methanocaldococcus ,Protein Folding ,Hot Temperature ,Archaeal Proteins ,Protein subunit ,Biophysics ,Gene Expression ,Crystallography, X-Ray ,Clostridium cellulolyticum ,Biochemistry ,Structural Biology ,TIM barrel ,Escherichia coli ,Genetics ,Structural Communications ,biology ,Active site ,Methanocaldococcus jannaschii ,Condensed Matter Physics ,biology.organism_classification ,Deoxyribonuclease IV (Phage T4-Induced) ,Recombinant Proteins ,Protein Structure, Tertiary ,Open reading frame ,Agrobacterium tumefaciens ,Structural Homology, Protein ,biology.protein ,Protein folding ,Protein Multimerization ,Carbohydrate Epimerases - Abstract
The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeonMethanocaldococcus jannaschiiwas determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase fromPseudomonas cichoriiand the D-psicose 3-epimerases fromAgrobacterium tumefaciensandClostridium cellulolyticum. However, the situation at the subunit–subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV.
- Published
- 2014
18. Structural insight into glucose dehydrogenase from the thermoacidophilic archaeonThermoplasma volcanium
- Author
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Kazunari Yoneda, Seiichiroh Uehara, Toshihisa Ohshima, Haruhiko Sakuraba, Yoshitaka Kanoh, and Hideyuki Iwata
- Subjects
Models, Molecular ,Protein Conformation ,Thermoplasma ,Thermoplasma volcanium ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Biology ,Crystallography, X-Ray ,Substrate Specificity ,chemistry.chemical_compound ,Structural Biology ,Glucose dehydrogenase ,Oxidoreductase ,Ternary complex ,chemistry.chemical_classification ,Binding Sites ,Nicotinic acid adenine dinucleotide phosphate ,Base Sequence ,ved/biology ,Sulfolobus solfataricus ,Substrate (chemistry) ,Glucose 1-Dehydrogenase ,General Medicine ,Glucose ,Enzyme ,chemistry ,Biochemistry ,Mutation ,NADP - Abstract
Glucose dehydrogenase from the thermoacidophilic archaeonThermoplasma volcanium(tvGlcDH) is highly active towards D-glucose and D-galactose, but does not utilize aldopentoses such as D-xylose as substrates. In the present study, the crystal structures of substrate/cofactor-free tvGlcDH and of a tvGlcDH T277F mutant in a binary complex with NADP and in a ternary complex with D-glucose and nicotinic acid adenine dinucleotide phosphate, an NADP analogue, were determined at resolutions of 2.6, 2.25 and 2.33 Å, respectively. The overall structure of each monomer showed notable similarity to that of the enzyme fromSulfolobus solfataricus(ssGlcDH-1), which accepts a broad range of C5 and C6 sugars as substrates. However, the amino-acid residues of tvGlcDH involved in substrate binding markedly differed from those of ssGlcDH-1. Structural comparison revealed that a decreased number of interactions between the C3-hydroxyl group of the sugar and the enzyme are likely to be responsible for the lack of reactivity of tvGlcDH towards D-xylose.
- Published
- 2014
19. The tertiary structure of an i-type lysozyme isolated from the common orient clam (Meretrix lusoria)
- Author
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Kazunari Yoneda, Tomohiro Araki, Yuya Kawaguchi, and Yuko Kuwano
- Subjects
animal structures ,Molecular Sequence Data ,Biophysics ,Biology ,Crystallography, X-Ray ,Biochemistry ,chemistry.chemical_compound ,Residue (chemistry) ,Structural Biology ,Aspartic acid ,Hydrolase ,Genetics ,Structural Communications ,Animals ,Amino Acid Sequence ,chemistry.chemical_classification ,Binding Sites ,Condensed Matter Physics ,biology.organism_classification ,Protein tertiary structure ,Bivalvia ,Protein Structure, Tertiary ,Amino acid ,Enzyme ,chemistry ,Muramidase ,Lysozyme ,Meretrix lusoria ,Chickens ,Sequence Alignment - Abstract
To evaluate the structure-function relationships of invertebrate lysozymes, a new invertebrate-type (i-type) lysozyme was isolated from the common orient clam (Meretrix lusoria) and the tertiary structure of this enzyme was determined. Comparison of the tertiary structure of this enzyme with those of chicken and Venerupi philippinarum lysozymes revealed that the location of the side chain of the second catalytic residue, an aspartic acid, and the N-acetylglucosamine trimer bound at subsites A-C were different. Furthermore, the amino acid electrostatically interacting with Asp30 in V. philippinarum lysozyme, Lys108, was substituted by Gly in M. lusoria lysozyme and no other possible amino acid that could contribute to this interaction was found in M. lusoria lysozyme. It therefore seems that the substitutions of the amino acids at the interface of the V. philippinarum lysozyme dimer are likely to change the oligomeric state of the M. lusoria lysozyme.
- Published
- 2013
20. Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis
- Author
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Junji, Hayashi, Kaori, Yamamoto, Kazunari, Yoneda, Toshihisa, Ohshima, and Haruhiko, Sakuraba
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Models, Molecular ,Binding Sites ,Archaeal Proteins ,Amino Acid Motifs ,Coenzymes ,Gene Expression ,Glycerolphosphate Dehydrogenase ,Hydrogen Bonding ,Crystallography, X-Ray ,NAD ,Protein Structure, Secondary ,Recombinant Proteins ,Kinetics ,Protein Subunits ,Escherichia coli ,Pyrobaculum ,Thermodynamics ,Protein Interaction Domains and Motifs ,Cloning, Molecular ,Protein Multimerization ,NADP ,Protein Binding - Abstract
A gene encoding an sn-glycerol-1-phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V
- Published
- 2016
21. Structure of a UDP-glucose dehydrogenase from the hyperthermophilic archaeonPyrobaculum islandicum
- Author
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Kazunari Yoneda, Toshihisa Ohshima, Haruhiko Sakuraba, and Tomoyuki Kawai
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Models, Molecular ,Biophysics ,Dehydrogenase ,Biology ,Uridine Diphosphate Glucose Dehydrogenase ,Biochemistry ,Substrate Specificity ,Protein structure ,Structural Biology ,Oxidoreductase ,Enzyme Stability ,Genetics ,Structural Communications ,Protein Interaction Domains and Motifs ,Protein Structure, Quaternary ,chemistry.chemical_classification ,Binding Sites ,Pyrobaculum ,Condensed Matter Physics ,biology.organism_classification ,Hyperthermophile ,carbohydrates (lipids) ,Enzyme ,chemistry ,NAD+ kinase ,Binding domain - Abstract
The crystal structure of an extremely thermostable UDP-glucose dehydrogenase (UDP-GDH) from the hyperthermophilic archaeon Pyrobaculum islandicum was determined at a resolution of 2.0 Å. The overall fold was comprised of an N-terminal NAD(+) dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long α-helix, and the main-chain coordinates of the enzyme were similar to those of previously studied UDP-GDHs, including the enzymes from Burkholderia cepacia, Streptococcus pyogenes and Klebsiella pneumoniae. However, the sizes of several surface loops in P. islandicum UDP-GDH were much smaller than the corresponding loops in B. cepacia UDP-GDH but were comparable to those of the S. pyogenes and K. pneumoniae enzymes. Structural comparison revealed that the presence of extensive intersubunit hydrophobic interactions, as well as the formation of an intersubunit aromatic pair network, is likely to be the main factor contributing to the hyperthermostability of P. islandicum UDP-GDH.
- Published
- 2012
22. Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium
- Author
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Kazunari Yoneda, Tomohiro Araki, Toshihisa Ohshima, and Haruhiko Sakuraba
- Subjects
Threonine ,Thermoplasma ,Archaeal Proteins ,Thermoplasma volcanium ,Dehydrogenase ,Biology ,Flavobacterium ,Biochemistry ,Substrate Specificity ,UDPglucose 4-Epimerase ,Oxidoreductase ,L-threonine dehydrogenase ,Escherichia coli ,Molecular replacement ,Molecular Biology ,Thermostability ,chemistry.chemical_classification ,Crystallography ,Cell Biology ,NAD ,biology.organism_classification ,carbohydrates (lipids) ,Alcohol Oxidoreductases ,chemistry ,Protein Structure and Folding ,Mutagenesis, Site-Directed ,NAD+ kinase - Abstract
A gene from the thermophilic archaeon Thermoplasma volcanium encoding an L-threonine dehydrogenase (L-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr(137) was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: L-ThrDH-NAD(+), L-ThrDH in complex with NAD(+) and pyruvate, Y137F mutant in complex with NAD(+) and L-threonine, and Y137F in complex with NAD(+) and L-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like L-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via direct proton transfer driven by Tyr(137). The factors contributing to the thermostability of T. volcanium L-ThrDH were analyzed by comparing its structure to that of F. frigidimaris L-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium L-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like L-ThrDH.
- Published
- 2012
23. Crystal structure of UDP-galactose 4-epimerase from the hyperthermophilic archaeon Pyrobaculum calidifontis
- Author
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Tomoyuki Kawai, Kazunari Yoneda, Toshihisa Ohshima, and Haruhiko Sakuraba
- Subjects
Models, Molecular ,Hot Temperature ,Protein Conformation ,Archaeal Proteins ,Protein subunit ,Molecular Sequence Data ,Static Electricity ,Biophysics ,Isomerase ,Crystallography, X-Ray ,Biochemistry ,Biophysical Phenomena ,Cofactor ,UDPglucose 4-Epimerase ,chemistry.chemical_compound ,Protein structure ,Catalytic Domain ,Enzyme Stability ,Ribose ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,Sequence Deletion ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,NAD ,Recombinant Proteins ,Hyperthermophile ,Protein Structure, Tertiary ,Molecular Weight ,carbohydrates (lipids) ,DNA, Archaeal ,chemistry ,Structural Homology, Protein ,Mutagenesis, Site-Directed ,Pyrobaculum ,biology.protein ,Mutant Proteins ,NAD+ kinase - Abstract
The crystal structure of a highly thermostable UDP-galactose 4-epimerase (GalE) from the hyperthermophilic archaeon Pyrobaculum calidifontis was determined at a resolution of 1.8Å. The asymmetric unit contained one subunit, and the functional dimer was generated by a crystallographic two-fold axis. Each monomer consisted of a Rossmann-fold domain with NAD bound and a carboxyl terminal domain. The overall structure of P. calidifontis GalE showed significant similarity to the structures of the GalEs from Escherichia coli, human and Trypanosoma brucei. However, the sizes of several surface loops were markedly smaller in P. calidifontis GalE than the corresponding loops in the other enzymes. Structural comparison revealed that the presence of an extensive hydrophobic interaction at the subunit interface is likely the main factor contributing to the hyperthermostability of the P. calidifontis enzyme. Within the NAD-binding site of P. calidifontis GalE, a loop (NAD-binding loop) tightly holds the adenine ribose moiety of NAD. Moreover, a deletion mutant lacking this loop bound NAD in a loose, reversible manner. Thus the presence of the NAD-binding loop in GalE is largely responsible for preventing the release of the cofactor from the holoenzyme.
- Published
- 2011
24. Crystal structure of UDP-galactose 4-epimerase-like l-threonine dehydrogenase belonging to the intermediate short-chain dehydrogenase-reductase superfamily
- Author
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Tadao Oikawa, Toshihisa Ohshima, Haruhiko Sakuraba, Kazunari Yoneda, and Ikuo Muraoka
- Subjects
chemistry.chemical_classification ,Short-chain dehydrogenase ,biology ,Stereochemistry ,Active site ,Dehydrogenase ,Cell Biology ,Substrate analog ,Biochemistry ,carbohydrates (lipids) ,NAD binding ,Crystallography ,chemistry.chemical_compound ,chemistry ,Oxidoreductase ,L-threonine dehydrogenase ,biology.protein ,NAD+ kinase ,Molecular Biology - Abstract
The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.
- Published
- 2010
25. First Crystal Structure of l-Lysine 6-Dehydrogenase as an NAD-dependent Amine Dehydrogenase
- Author
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Kazunari Yoneda, Haruhiko Sakuraba, Junya Fukuda, and Toshihisa Ohshima
- Subjects
Rossmann fold ,Hot Temperature ,Protein subunit ,Molecular Sequence Data ,Coenzymes ,Dehydrogenase ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Protein Structure, Secondary ,Pyrococcus horikoshii ,Catalytic Domain ,Amino Acid Sequence ,Protein Structure, Quaternary ,Molecular Biology ,biology ,Chemistry ,Lysine ,Amine dehydrogenase ,Active site ,Saccharopine dehydrogenase ,Cell Biology ,NAD ,biology.organism_classification ,Recombinant Proteins ,Protein Structure and Folding ,Mutagenesis, Site-Directed ,biology.protein ,Saccharopine Dehydrogenases ,NAD+ kinase ,Oxidoreductases - Abstract
A gene encoding an L-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable L-lysine dehydrogenase yet described, with a half-life of 180 min at 100 degrees C. The product of the enzyme's catalytic activity is Delta(1)-piperideine-6-carboxylate, which makes this enzyme an L-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the epsilon- amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 A in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of l-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase.
- Published
- 2010
26. Structure of a<scp>D</scp>-tagatose 3-epimerase-related protein from the hyperthermophilic bacteriumThermotoga maritima
- Author
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Haruhiko Sakuraba, Ryushi Kawakami, Toshihisa Ohshima, Kazunari Yoneda, and Takenori Satomura
- Subjects
Biophysics ,Isomerase ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,chemistry.chemical_compound ,Protein structure ,Bacterial Proteins ,Structural Biology ,Catalytic Domain ,Genetics ,Structural Communications ,Thermotoga maritima ,Hexoses ,Pseudomonas cichorii ,biology ,Agrobacterium tumefaciens ,Condensed Matter Physics ,biology.organism_classification ,Hyperthermophile ,Open reading frame ,chemistry ,Structural Homology, Protein ,Carbohydrate Epimerases ,Ketohexose - Abstract
The crystal structure of a D-tagatose 3-epimerase-related protein (TM0416p) encoded by the hypothetical open reading frame TM0416 in the genome of the hyperthermophilic bacterium Thermotoga maritima was determined at a resolution of 2.2 A. The asymmetric unit contained two homologous subunits and a dimer was generated by twofold symmetry. The main-chain coordinates of the enzyme monomer proved to be similar to those of D-tagatose 3-epimerase from Pseudomonas cichorii and D-psicose 3-epimerase from Agrobacterium tumefaciens; however, TM0416p exhibited a unique solvent-accessible substrate-binding pocket that reflected the absence of an alpha-helix that covers the active-site cleft in the two aforementioned ketohexose 3-epimerases. In addition, the residues responsible for creating a hydrophobic environment around the substrate in TM0416p differ entirely from those in the other two enzymes. Collectively, these findings suggest that the substrate specificity of TM0416p is likely to differ substantially from those of other D-tagatose 3-epimerase family enzymes.
- Published
- 2009
27. Structure of l-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii
- Author
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Issaku Asai, Haruhiko Sakuraba, Kazunari Yoneda, Hideaki Tsuge, Toshihisa Ohshima, and Nobuhiko Katunuma
- Subjects
Hot Temperature ,Protein Conformation ,Archaeal Proteins ,Molecular Sequence Data ,Biophysics ,Sulfolobus tokodaii ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Sulfolobus ,Analytical Chemistry ,Bacterial Proteins ,Oxidoreductase ,Escherichia coli ,medicine ,Amino Acid Sequence ,L-aspartate oxidase ,Molecular Biology ,Thermostability ,chemistry.chemical_classification ,Escherichia coli Proteins ,biology.organism_classification ,Recombinant Proteins ,Hyperthermophile ,Crystallography ,chemistry ,Flavin-Adenine Dinucleotide ,Amino Acid Oxidoreductases ,Linker ,Archaea - Abstract
The crystal structure of the highly thermostable l -aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 A resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an α + β capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a β-strand that runs parallel to the C-terminal strand. This results in the formation of an additional β-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO.
- Published
- 2008
28. Sequential Aldol Condensation Catalyzed by Hyperthermophilic 2-Deoxy- <scp>d</scp> -Ribose-5-Phosphate Aldolase
- Author
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Kazunari Yoneda, Kyoko Satoh, Hitoshi Hori, Toshihisa Ohshima, Hideaki Tsuge, Yoshihiro Uto, Kumiko Yoshihara, Haruhiko Sakuraba, Katsuyuki Takahashi, and Ryushi Kawakami
- Subjects
Models, Molecular ,Hot Temperature ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Archaeal Proteins ,Acetaldehyde ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Catalysis ,Protein Structure, Secondary ,Substrate Specificity ,Enzyme catalysis ,Structure-Activity Relationship ,Enzyme Stability ,medicine ,Thermotoga maritima ,Enzymology and Protein Engineering ,Escherichia coli ,Aldehyde-Lyases ,chemistry.chemical_classification ,Binding Sites ,Molecular Structure ,Ecology ,biology ,Pyrobaculum ,Aldolase A ,Hydrogen-Ion Concentration ,biology.organism_classification ,Lyase ,Hyperthermophile ,Protein Structure, Tertiary ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,Ribosemonophosphates ,Food Science ,Biotechnology - Abstract
Genes encoding 2-deoxy- d -ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima , were expressed individually in Escherichia coli , after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25°C), although both enzymes showed much less 2-deoxy- d -ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25°C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 Å. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.
- Published
- 2007
29. Crystal structure of archaeal highly thermostable<scp>L</scp>-aspartate dehydrogenase/NAD/citrate ternary complex
- Author
-
Kazunari Yoneda, Hideaki Tsuge, Toshihisa Ohshima, Nobuhiko Katunuma, and Haruhiko Sakuraba
- Subjects
chemistry.chemical_classification ,biology ,Chemistry ,Active site ,Dehydrogenase ,Cell Biology ,Substrate analog ,Thermotoga ,biology.organism_classification ,Biochemistry ,Crystallography ,chemistry.chemical_compound ,Oxidoreductase ,biology.protein ,Coenzyme binding ,NAD+ kinase ,Molecular Biology ,Ternary complex - Abstract
The crystal structure of the highly thermostable l-aspartate dehydrogenase (l-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidusl-aspDH was refined at a resolution of 1.9 A with a crystallographic R-factor of 21.7% (Rfree = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidusl-aspDH/NAD/citrate ternary complex and the Thermotoga maritimal-aspDH/NAD binary complex showed that A. fulgidusl-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritimal-aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidusl-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two l-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to l-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic l-aspDH.
- Published
- 2007
30. Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases
- Author
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Kazunari Yoneda, Kwang Kim, Yutaka Kawarabayasi, Haruhiko Sakuraba, Junji Hayashi, Shota Inoue, and Toshihisa Ohshima
- Subjects
Models, Molecular ,Protein Conformation ,Molecular Sequence Data ,Sequence alignment ,Plasma protein binding ,Article ,Catalysis ,Substrate Specificity ,Protein structure ,Oxidoreductase ,Homoserine Dehydrogenase ,Amino Acid Sequence ,Binding site ,chemistry.chemical_classification ,Homoserine dehydrogenase ,Cofactor binding ,Multidisciplinary ,Binding Sites ,biology ,biology.organism_classification ,Archaea ,Kinetics ,Biochemistry ,chemistry ,Oxidoreductases ,Sequence Alignment ,NADP ,Protein Binding - Abstract
NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.
- Published
- 2015
31. A novel NAD(P)H-dependent carbonyl reductase specifically expressed in the thyroidectomized chicken fatty liver: catalytic properties and crystal structure
- Author
-
Toshihisa Ohshima, Tomohiro Araki, Haruhiko Sakuraba, Takeshi Shibata, Kazunari Yoneda, Yudai Fukuda, and Takeki Sone
- Subjects
7-Dehydrocholesterol reductase ,Carbonyl Reductase ,Stereochemistry ,Protein Conformation ,Molecular Sequence Data ,Aldo-Keto Reductases ,Reductase ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Hypothyroidism ,Aldehyde Reductase ,Catalytic Domain ,Animals ,Enzyme kinetics ,Amino Acid Sequence ,Databases, Protein ,Molecular Biology ,biology ,Chemistry ,Protein Stability ,Active site ,Substrate (chemistry) ,Cell Biology ,Phenanthrenes ,Recombinant Proteins ,Fatty Liver ,Disease Models, Animal ,Protein Subunits ,Amino Acid Substitution ,Liver ,biology.protein ,Biocatalysis ,Tyrosine ,Mutant Proteins ,NAD+ kinase ,Chickens ,NADP - Abstract
A gene encoding a functionally unknown protein that is specifically expressed in the thyroidectomized chicken fatty liver and has a predicted amino acid sequence similar to that of NAD(P)H-dependent carbonyl reductase was overexpressed in Escherichia coli; its product was purified and characterized. The expressed enzyme was an NAD(P)H-dependent broad substrate specificity carbonyl reductase and was inhibited by arachidonic acid at 1.5 μm. Enzymological characterization indicated that the enzyme could be classified as a cytosolic-type carbonyl reductase. The enzyme's 3D structure was determined using the molecular replacement method at 1.98 A resolution in the presence of NADPH and ethylene glycol. The asymmetric unit consisted of two subunits, and a noncrystallographic twofold axis generated the functional dimer. The structures of the subunits, A and B, differed from each other. In subunit A, the active site contained an ethylene glycol molecule absent in subunit B. Consequently, Tyr172 in subunit A rotated by 103.7° in comparison with subunit B, which leads to active site closure in subunit A. In Y172A mutant, the Km value for 9,10-phenanthrenequinone (model substrate) was 12.5 times higher than that for the wild-type enzyme, indicating that Tyr172 plays a key role in substrate binding in this carbonyl reductase. Because the Tyr172-containing active site lid structure (Ile164-Gln174) is not conserved in all known carbonyl reductases, our results provide new insights into substrate binding of carbonyl reductase. The catalytic properties and crystal structure revealed that thyroidectomized chicken fatty liver carbonyl reductase is a novel enzyme.
- Published
- 2015
32. The first archaeal l-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: Gene cloning and enzymological characterization
- Author
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Kazunari Yoneda, Shuichiro Goda, Ryushi Kawakami, Haruhiko Sakuraba, Toshihisa Ohshima, and Yuya Tagashira
- Subjects
Oxaloacetic Acid ,Molecular Sequence Data ,Biophysics ,Dehydrogenase ,Biochemistry ,Analytical Chemistry ,Enzyme Stability ,Escherichia coli ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Aspartate dehydrogenase ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,biology ,Archaeoglobus fulgidus ,biology.organism_classification ,Hyperthermophile ,Protein Structure, Tertiary ,Thermococcales ,Kinetics ,Enzyme ,chemistry ,Amino Acid Oxidoreductases ,NAD+ kinase ,Sulfolobales ,Dimerization - Abstract
A gene encoding an l -aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus . After expression in Escherichia coli , the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable l -aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 °C. The optimum temperature for l -aspartate dehydrogenation was about 80 °C. The enzyme specifically utilized l -aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The K m values for l -aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The K m values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the K m values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro- R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal l -aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.
- Published
- 2006
33. Functional and structural characteristics of methylmalonyl-CoA mutase from Pyrococcus horikoshii
- Author
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Haruhiko Sakuraba, Yukinori Yabuta, Toshihisa Ohshima, Yoshihisa Nakano, Yukiko Kamei, Jiro Arima, Kazunari Yoneda, Tomohiro Bito, and Fumio Watanabe
- Subjects
Immunoprecipitation ,Molecular Sequence Data ,Applied Microbiology and Biotechnology ,Biochemistry ,Cofactor ,Analytical Chemistry ,law.invention ,Pyrococcus horikoshii ,Mutase ,law ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Gene ,biology ,Sequence Homology, Amino Acid ,Organic Chemistry ,Methylmalonyl-CoA mutase ,Methylmalonyl-CoA Mutase ,General Medicine ,biology.organism_classification ,Recombinant Proteins ,biology.protein ,Recombinant DNA ,Electrophoresis, Polyacrylamide Gel ,Cobamides ,Protein Multimerization ,Biotechnology ,Archaea - Abstract
Methylmalonyl-CoA mutase (MCM) requires 5′-deoxyadenosylcobalamin (AdoCbl) as a cofactor and is widely distributed in organisms from bacteria and animals. Although genes encoding putative MCMs are present in many archaea, they are separately encoded in large and small subunits. The large and small subunits of archaeal MCM are similar to the catalytic and AdoCbl-binding domains of human MCM, respectively. In Pyrococcus horikoshii OT3, putative genes PH1306 and PH0275 encode the large and small subunits, respectively. Because information on archaeal MCM is extremely restricted, we examined the functional and structural characteristics of P. horikoshii MCM. Reconstitution experiments using recombinant PH0275 and PH1306 showed that these proteins assemble in equimolar ratios and form of heterotetrameric complexes in the presence of AdoCbl. Subsequent immunoprecipitation experiments using anti-PH0275 and anti-PH1306 antibodies suggested that PH0275 and PH1306 form a complex in P. horikoshii cells in the presence of AdoCbl.
- Published
- 2014
34. Crystal Structure of the NAD Biosynthetic Enzyme Quinolinate Synthase
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Toshihisa Ohshima, Hideaki Tsuge, Nobuhiko Katunuma, Kazunari Yoneda, and Haruhiko Sakuraba
- Subjects
Protein Conformation ,Stereochemistry ,Archaeal Proteins ,Malates ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Phosphates ,Pyrococcus horikoshii ,chemistry.chemical_compound ,Protein structure ,Multienzyme Complexes ,Escherichia coli ,Cloning, Molecular ,Molecular Biology ,Dihydroxyacetone phosphate ,chemistry.chemical_classification ,Binding Sites ,ATP synthase ,biology ,Active site ,Cell Biology ,NAD ,biology.organism_classification ,Quinolinate ,Crystallography ,Enzyme ,chemistry ,Structural Homology, Protein ,biology.protein ,NAD+ kinase - Abstract
A gene encoding a quinolinate synthase has been identified in the hyperthermophilic archaeon Pyrococcus horikoshii via genome sequencing. The gene was overexpressed in Escherichia coli, and the crystal structure of the produced enzyme was determined to 2.0 A resolution in the presence of malate, a substrate analogue. The overall structure exhibits a unique triangular architecture composed of a 3-fold repeat of three-layer (alphabetaalpha) sandwich folding. Although some aspects of the fold homologous to the each domain have been observed previously, the overall structure of quinolinate synthase shows no similarity to any known protein structure. The three analogous domains are related to a pseudo-3-fold symmetry. The active site is located at the interface of the three domains and is centered on the pseudo-3-fold axis. The malate molecule is tightly held near the bottom of the active site cavity. The model of the catalytic state during the first condensation step of the quinolinate synthase reaction indicates that the elimination of inorganic phosphate from dihydroxyacetone phosphate may precede the condensation reaction.
- Published
- 2005
35. Expression, purification, crystallization and preliminary X-ray analysis of NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver
- Author
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Toshihisa Ohshima, Kazunari Yoneda, Takeshi Shibata, Haruhiko Sakuraba, Yudai Fukuda, Tomohiro Araki, and Takahiro Nikki
- Subjects
animal structures ,Carbonyl Reductase ,Stereochemistry ,Biophysics ,Alcohol oxidoreductase ,Polyethylene glycol ,Crystallography, X-Ray ,Biochemistry ,Polyethylene Glycols ,law.invention ,chemistry.chemical_compound ,Structural Biology ,law ,Genetics ,medicine ,Animals ,Crystallization ,Chemistry ,Fatty liver ,Space group ,Condensed Matter Physics ,medicine.disease ,Fatty Liver ,Alcohol Oxidoreductases ,Crystallization Communications ,Thyroidectomy ,NAD+ kinase ,Chickens ,NADP ,Monoclinic crystal system - Abstract
An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 300 as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.26, b = 81.32, c = 77.27 A, β = 119.43°, and diffracted to 1.86 A resolution on beamline NE3A at the Photon Factory. The overall R merge was 5.4% and the data completeness was 99.4%.
- Published
- 2012
36. The structure of the proteinaceous inhibitor PliI from Aeromonas hydrophila in complex with its target lysozyme
- Author
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Kazunari Yoneda, Joris M. Van Herreweghe, Makoto Ogata, Tomohiro Araki, Christiaan Michiels, Seppe Leysen, Sergei V. Strelkov, and Taichi Usui
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Models, Molecular ,Protein Conformation ,Mutant ,Molecular Sequence Data ,Crystallography, X-Ray ,chemistry.chemical_compound ,Functional importance ,Bacterial Proteins ,Structural Biology ,Animals ,Amino Acid Sequence ,biology ,General Medicine ,Periplasmic space ,biology.organism_classification ,Aeromonas hydrophila ,Bivalvia ,Biochemistry ,chemistry ,Lytic cycle ,Muramidase ,Lysozyme ,Meretrix lusoria ,Gram-Negative Bacterial Infections ,Sequence Alignment ,Bacteria - Abstract
Recent microbiological data have revealed that Gram-negative bacteria are able to protect themselves against the lytic action of host lysozymes by secreting proteinaceous inhibitors. Four distinct classes of such inhibitors have been discovered that specifically act against c-type, g-type and i-type lysozymes. Here, the 1.24 Å resolution crystal structure of the periplasmic i-type lysozyme inhibitor from Aeromonas hydrophila (PliI-Ah) in complex with the i-type lysozyme from Meretrix lusoria is reported. The structure is the first to explain the inhibitory mechanism of the PliI family at the atomic level. A distinct `ridge' formed by three exposed PliI loops inserts into the substrate-binding groove of the lysozyme, resulting in a complementary `key-lock' interface. The interface is principally stabilized by the interactions made by the PliI-Ah residues Ser104 and Tyr107 belonging to the conserved SGxY motif, as well as by the other conserved residues Ser46 and Asp76. The functional importance of these residues is confirmed by inhibition assays with the corresponding point mutants of PliI-Ah. The accumulated structural data on lysozyme-inhibitor complexes from several classes indicate that in all cases an extensive interface of either a single or a double `key-lock' type is formed, resulting in highly efficient inhibition. These data provide a basis for the rational development of a new class of antibacterial drugs. journal: Acta Crystallographica Section D: Biological Crystallography content_type: research papers peer_reviewed: Yes review_process: Single blind received: 2 July 2014 accepted: 26 November 2014 published_online: 23 January 2015 supplementary_materials: This article has supporting information copyright: © 2015 International Union of Crystallography ispartof: Acta Crystallographica D vol:71 issue:Pt 2 pages:344-351 ispartof: location:United States status: published
- Published
- 2014
37. The complete amino acid sequence and enzymatic properties of an i-type lysozyme isolated from the common orient clam (Meretrix lusoria)
- Author
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Kazunari Yoneda, Yuya Kawaguchi, Tomohiro Araki, Norie Araki, and Yuko Kuwano
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Glycosylation ,Dimer ,Molecular Sequence Data ,Applied Microbiology and Biotechnology ,Biochemistry ,Isozyme ,Analytical Chemistry ,Substrate Specificity ,chemistry.chemical_compound ,Venerupis philippinarum ,Enzyme Stability ,Animals ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Phylogeny ,chemistry.chemical_classification ,biology ,Organic Chemistry ,Osmolar Concentration ,General Medicine ,Sequence Analysis, DNA ,Hydrogen-Ion Concentration ,biology.organism_classification ,Amino acid ,Bivalvia ,Isoenzymes ,Kinetics ,Enzyme ,chemistry ,Muramidase ,Lysozyme ,Meretrix lusoria ,Chickens ,Biotechnology - Abstract
To determine the structure and functional relationships of invertebrate lysozymes, we isolated a new invertebrate (i)-type lysozyme from the common orient clam (Meretrix lusoria) and determined the complete amino acid sequence of two isozymes that differed by one amino acid. The determined sequence showed 65% similarity to a lysozyme from Venerupis philippinarum (Tapes japonica), and it was therefore classified as an i-type lysozyme. The lytic activities of this lysozyme were similar to those of previously reported bivalve i-type lysozymes, but unlike the V. philippinarum lysozyme, it did not exhibit an increase in activity in high ionic strength. Our data suggest that this lysozyme does not have a dimeric structure, due to the replacement of Lys108 which contributes to dimer formation in the V. philippinarum lysozyme. GlcNAc oligomer activities suggested an absence of transglycosylation activity and a higher number of subsites on this enzyme compared with hen egg lysozyme.
- Published
- 2013
38. Functional and structural characteristics of methylmalonyl-CoA mutase from Pyrococcus horikoshii
- Author
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Yukinori Yabuta, Yukiko Kamei, Tomohiro Bito, Jiro Arima, Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, Yoshihisa Nakano, Fumio Watanabe, Yukinori Yabuta, Yukiko Kamei, Tomohiro Bito, Jiro Arima, Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, Yoshihisa Nakano, and Fumio Watanabe
- Published
- 2015
- Full Text
- View/download PDF
39. Molecular cloning and expression of α-globin and β-globin genes from crocodile (Crocodylus siamensis)
- Author
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Preeyanan Anwised, Theeranan Temsiripong, Apisak Dhiravisit, Sompong Thammasirirak, Sarawut Jitrapakdee, Tomohiro Araki, Kazunari Yoneda, and Thai Kabbua
- Subjects
Molecular Sequence Data ,Gene Expression ,Bioengineering ,Reptilian Proteins ,beta-Globins ,Molecular cloning ,Biochemistry ,Analytical Chemistry ,law.invention ,Rapid amplification of cDNA ends ,alpha-Globins ,law ,Complementary DNA ,Animals ,Humans ,Globin ,Amino Acid Sequence ,Cloning, Molecular ,Phylogeny ,Alligators and Crocodiles ,biology ,Base Sequence ,Organic Chemistry ,biology.organism_classification ,Molecular biology ,Open reading frame ,Crocodylus siamensis ,Vertebrates ,Recombinant DNA ,Heterologous expression ,Sequence Alignment - Abstract
The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.
- Published
- 2013
40. Crystallization and preliminary X-ray analysis of l-serine 3-dehydrogenase complexed with NADP+ from the hyperthermophilic archaeon Pyrobaculum calidifontis
- Author
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Haruhiko Sakuraba, Kazunari Yoneda, Takahiro Nikki, Tomohiro Araki, Toshihisa Ohshima, and Takeshi Shibata
- Subjects
Ammonium sulfate ,genetic processes ,Biophysics ,information science ,Dehydrogenase ,Crystallography, X-Ray ,Biochemistry ,law.invention ,Serine ,chemistry.chemical_compound ,Structural Biology ,law ,Genetics ,Crystallization ,biology ,Pyrobaculum ,Condensed Matter Physics ,biology.organism_classification ,Hyperthermophile ,Crystallography ,enzymes and coenzymes (carbohydrates) ,chemistry ,Crystallization Communications ,biological sciences ,health occupations ,Electrophoresis, Polyacrylamide Gel ,NAD+ kinase ,Oxidoreductases ,NADP ,Monoclinic crystal system - Abstract
An NAD(P)(+)-dependent L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 120.81, b = 57.40, c = 56.37 Å, β = 106.88°. Diffraction data were collected to 1.57 Å resolution on beamline NE3A at the Photon Factory. The overall R(merge) was 4.2% and the data completeness was 90.1%.
- Published
- 2013
41. Crystallization and preliminary X-ray diffraction analysis of the hyperthermostable NAD-dependent glutamate dehydrogenase fromPyrobaculum islandicum
- Author
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Kazunari Yoneda, Toshihisa Ohshima, Haruhiko Sakuraba, Mohammad W. Bhuiya, Takahito Imagawa, Nobuhiko Katunuma, and Hideaki Tsuge
- Subjects
Archaeal Proteins ,Glutamate dehydrogenase ,Resolution (electron density) ,General Medicine ,Random hexamer ,Crystallography, X-Ray ,law.invention ,Solvent ,chemistry.chemical_compound ,Crystallography ,chemistry ,Structural Biology ,law ,Enzyme Stability ,X-ray crystallography ,Orthorhombic crystal system ,Cloning, Molecular ,Crystallization ,Glutamate Dehydrogenase (NADP+) ,Derivative (chemistry) - Abstract
NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum was crystallized in the apo- and holoenzyme forms. Crystals were obtained using 2-propanol and polyethylene glycol MME 550 as precipitants for the apoenzyme and holoenzyme, respectively. The apoenzyme crystals belong to the trigonal space group P3(1)21 or its enantiomorph P3(2)21. The asymmetric unit contains three subunits; the values of the Matthews coefficient (VM) and the solvent content are 2.9 A3 Da-1 and 57%, respectively. A native data set was collected to a highest resolution limit of 4.0 A on an in-house X-ray source using a rotating-anode generator (overall Rsym of 12.3% and completeness of 97%). The holoenzyme crystals belong to the orthorhombic space group P2(1)2(1)2(1); the asymmetric unit contains one hexamer, giving a VM of 2.79 A3 Da-1 and a solvent content of 55%. Native and derivative data sets were collected. The crystals diffract to a maximum resolution of 2.8 A on the KEK-NW12 beamline at the Photon Factory and gave a data set with an overall Rsym of 7.9% and a completeness of 91%. Attempts are being made to solve the structure by the SIRAS method.
- Published
- 2004
42. Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix
- Author
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Ryushi Kawakami, Takenori Satomura, Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, Yusuke Hara, and Kwang Kim
- Subjects
chemistry.chemical_classification ,Aeropyrum ,Archaeal Proteins ,Active site ,Dehydrogenase ,Cell Biology ,Biology ,biology.organism_classification ,Crystallography, X-Ray ,Biochemistry ,Heterotetramer ,Hyperthermophile ,Protein Structure, Tertiary ,Crystallography ,Proline dehydrogenase ,chemistry ,Oxidoreductase ,Protein Structure and Folding ,biology.protein ,Proline Oxidase ,Aeropyrum pernix ,Protein Multimerization ,Protein Structure, Quaternary ,Molecular Biology - Abstract
Two types of dye-linked L-proline dehydrogenase (PDH1, α4β4-type hetero-octamer, and PDH2, αβγδ-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 β-subunit, which is responsible for catalyzing L-proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu(428), which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an L-proline molecule was entrapped within the active site cavity. The K(m) value of a Leu(428) deletion mutant for L-proline was about 800 times larger than the K(m) value of the wild-type enzyme, although the k(cat) values did not differ much between the two enzymes. This suggests the C-terminal Leu(428) is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with L-proline bound, and it provides new insight into the substrate binding of LPDH.
- Published
- 2012
43. Structure of a multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum
- Author
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Kazunari Yoneda, Yasuhiro Kashima, Haruhiko Sakuraba, Kohtaroh Koga, and Toshihisa Ohshima
- Subjects
Models, Molecular ,Stereochemistry ,Pyrobaculum aerophilum ,Molecular Sequence Data ,Biophysics ,Bacillus subtilis ,medicine.disease_cause ,Multicopper oxidase ,Crystallography, X-Ray ,Biochemistry ,Structural Biology ,Oxidoreductase ,Enzyme Stability ,Genetics ,medicine ,Structural Communications ,Amino Acid Sequence ,Escherichia coli ,Conserved Sequence ,chemistry.chemical_classification ,biology ,Condensed Matter Physics ,biology.organism_classification ,Hyperthermophile ,Protein Structure, Tertiary ,chemistry ,Structural Homology, Protein ,Pyrobaculum ,Oxidoreductases ,Sequence Alignment ,Copper ,Archaea - Abstract
The crystal structure of an extremely thermostable multicopper oxidase (McoP) from the hyperthermophilic archaeon Pyrobaculum aerophilum was determined at a resolution of 2.0 A. The overall fold was comprised of three cupredoxin-like domains and the main-chain coordinates of the enzyme were similar to those of multicopper oxidases from Escherichia coli (CueO) and Bacillus subtilis (CotA). However, there were clear topological differences around domain 3 between McoP and the other two enzymes: a methionine-rich helix in CueO and a protruding helix in CotA were not present in McoP. Instead, a large loop (PL-1) covered the T1 copper centre of McoP and a short α-helix in domain 3 extended near the N-terminal end of PL-1. In addition, the sizes of several surface loops in McoP were markedly smaller than the corresponding loops in CueO and CotA. Structural comparison revealed that the presence of extensive hydrophobic interactions and a smaller cavity volume are likely to be the main factors contributing to the hyperthermostability of McoP.
- Published
- 2011
44. Crystal structure of UDP-galactose 4-epimerase-like L-threonine dehydrogenase belonging to the intermediate short-chain dehydrogenase-reductase superfamily
- Author
-
Kazunari, Yoneda, Haruhiko, Sakuraba, Ikuo, Muraoka, Tadao, Oikawa, and Toshihisa, Ohshima
- Subjects
Models, Molecular ,Threonine ,Alcohol Oxidoreductases ,UDPglucose 4-Epimerase ,Protein Conformation ,Catalytic Domain ,Escherichia coli ,Humans ,Crystallography, X-Ray ,Flavobacterium - Abstract
The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.
- Published
- 2010
45. Visible wavelength spectrophotometric assays of L-aspartate and D-aspartate using hyperthermophilic enzyme systems
- Author
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Toshihisa Ohshima, Taketo Ohmori, Katsumi Doi, Haruhiko Sakuraba, Yuta Mutaguchi, and Kazunari Yoneda
- Subjects
Swine ,Biophysics ,Dehydrogenase ,Aspartate racemase ,Nicotinamide adenine dinucleotide ,Biochemistry ,chemistry.chemical_compound ,Mice ,Isomerism ,Animals ,Molecular Biology ,Aspartate dehydrogenase ,Acetic Acid ,Amino Acid Isomerases ,chemistry.chemical_classification ,Oxidase test ,Aspartic Acid ,Chromatography ,Chemistry ,Escherichia coli Proteins ,D-Aspartic Acid ,Cell Biology ,NAD ,Enzyme ,Liver ,Spectrophotometry ,Methylphenazonium Methosulfate ,NAD+ kinase ,Amino Acid Oxidoreductases ,Formazan ,Oxidation-Reduction - Abstract
Methods with which to simply and rapidly assay L-aspartate (L-Asp) and D-aspartate (D-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of L- and D-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an L-aspartate dehydrogenase (L-AspDH) system to colorimetrically assay L-Asp and a system of three hyperthermophilic enzymes--aspartate racemase (AspR), L-AspDH, and L-aspartate oxidase (L-AO)--to assay D-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD(+))-dependent L-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, D-Asp was measured after first removing L-Asp in the sample solution with L-AO. The remaining D-Asp was then changed to L-Asp using racemase, and the newly formed L-Asp was assayed calorimetrically using NAD(+)-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM L- and D-Asp in the assay systems. In addition, methods were applicable to the L- and D-Asp determinations in some living cells and foods.
- Published
- 2010
46. Structure of the newly found green turtle egg-white ribonuclease
- Author
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Tomohiro Araki, Somporn Katekaew, Kazunari Yoneda, Yoshimitsu Kakuta, Makoto Kimura, Buabarn Kuaprasert, and Takao Torikata
- Subjects
Models, Molecular ,Angiogenin ,RNase P ,Molecular Sequence Data ,Biophysics ,Protein Data Bank (RCSB PDB) ,Crystallography, X-Ray ,Biochemistry ,RNase PH ,Ribonucleases ,Structural Biology ,Genetics ,Animals ,Structural Communications ,Ribonuclease ,Amino Acid Sequence ,Ovum ,chemistry.chemical_classification ,biology ,Condensed Matter Physics ,Molecular biology ,Protein Structure, Tertiary ,Turtles ,Enzyme ,chemistry ,Structural biology ,Structural Homology, Protein ,biology.protein ,Sequence Alignment ,Algorithms ,Egg white - Abstract
Marine green turtle (Chelonia mydas) egg-white ribonuclease (GTRNase) was crystallized from 1.1 M ammonium sulfate pH 5.5 and 30% glycerol using the sitting-drop vapour-diffusion method. The structure of GTRNase has been solved at 1.60 A resolution by the molecular-replacement technique using a model based on the structure of RNase 5 (murine angiogenin) from Mus musculus (46% identity). The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 86.271, b = 34.174, c = 39.738 A, alpha = 90, beta = 102, gamma = 90 degrees . GTRNase consists of three helices and seven beta-strands and displays the alpha+beta folding topology typical of a member of the RNase A superfamily. Superposition of the C(alpha) coordinates of GTRNase and RNase A superfamily members indicates that the overall structure is highly similar to that of angiogenin or RNase 5 from M. musculus (PDB code 2bwl) and RNase A from Bos taurus (PDB code 2blz), with root-mean-square deviations of 3.9 and 2.0 A, respectively. The catalytic residues are conserved with respect to the RNase A superfamily. The three disulfide bridges observed in the reptilian enzymes are conserved in GTRNase, while one further disulfide bond is required for the structural stability of mammalian RNases. GTRNase is expressed in egg white and the fact that its sequence has the highest similarity to that of snapping turtle pancreatic RNase suggests that the GTRNase secreted from oviduct cells to form egg white is probably the product of the same gene as activated in pancreatic cells.
- Published
- 2010
47. Catalytic properties and crystal structure of quinoprotein aldose sugar dehydrogenase from hyperthermophilic archaeon Pyrobaculum aerophilum
- Author
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Akira Watanabe, Takenori Satomura, Junko Motonaka, Kazunari Yoneda, Haruhiko Sakuraba, Kaori Yokono, Toshihisa Ohshima, Yasuhiko Asada, and Tomoki Yabutani
- Subjects
Glucose Dehydrogenases ,Biophysics ,Carbohydrates ,PQQ Cofactor ,Dehydrogenase ,Biology ,medicine.disease_cause ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Pyrroloquinoline quinone ,Glucose dehydrogenase ,Oxidoreductase ,medicine ,Escherichia coli ,Molecular Biology ,Quinoprotein glucose dehydrogenase ,chemistry.chemical_classification ,Binding Sites ,Archaea ,Hyperthermophile ,Enzyme ,chemistry ,Pyrobaculum ,Oxidoreductases - Abstract
We identified a gene encoding a soluble quinoprotein glucose dehydrogenase homologue in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The enzyme was extremely thermostable, and the activity of the pyrroloquinoline quinone (PQQ)-bound holoenzyme was not lost after incubation at 100 degrees C for 10 min. The crystal structure of the enzyme was determined in both the apoform and as the PQQ-bound holoenzyme. The overall fold of the P. aerophilum enzyme showed significant similarity to that of soluble quinoprotein aldose sugar dehydrogenase (Asd) from E. coli. However, clear topological differences were observed in the two long loops around the PQQ-binding sites of the two enzymes. Structural comparison revealed that the hyperthermostability of the P. aerophilum enzyme is likely attributable to the presence of an extensive aromatic pair network located around a beta-sheet involving N- and C-terminal beta-strands.
- Published
- 2010
48. Structure of an archaeal alanine:glyoxylate aminotransferase
- Author
-
Haruhiko Sakuraba, Hideaki Tsuge, Nobuhiko Katunuma, Kazunari Yoneda, Kiyo Takeuchi, and Toshihisa Ohshima
- Subjects
Models, Molecular ,Crystallography, X-Ray ,Substrate Specificity ,Tetramer ,Species Specificity ,Structural Biology ,Catalytic Domain ,Transferase ,Thermococcus litoralis ,Protein Structure, Quaternary ,Transaminases ,chemistry.chemical_classification ,Alanine ,biology ,Thermus thermophilus ,Substrate (chemistry) ,General Medicine ,biology.organism_classification ,Recombinant Proteins ,Protein Structure, Tertiary ,Thermococcus ,Enzyme ,Biochemistry ,chemistry ,Archaea - Abstract
The crystal structure of a novel alanine:glyoxylate aminotransferase from the hyperthermophilic archaeon Thermococcus litoralis was determined at 2.3 A resolution. The asymmetric unit contains four homologous subunits and the functional tetramer is generated by noncrystallographic 222 symmetry. Although the main-chain coordinates of the monomer of the Thermococcus litoralis enzyme showed a high degree of similarity to those of aspartate aminotransferase from Thermus thermophilus HB8, the amino-acid residues involved in substrate binding in the aspartate aminotransferase are only partially conserved in the Thermococcus litoralis enzyme. This may account for the difference in the substrate specificities of the two enzymes.
- Published
- 2007
49. Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase/NAD/citrate ternary complex
- Author
-
Kazunari, Yoneda, Haruhiko, Sakuraba, Hideaki, Tsuge, Nobuhiko, Katunuma, and Toshihisa, Ohshima
- Subjects
Models, Molecular ,Aspartic Acid ,Binding Sites ,Sequence Homology, Amino Acid ,Archaeal Proteins ,Molecular Sequence Data ,Hydrogen Bonding ,Crystallography, X-Ray ,NAD ,Citric Acid ,Protein Structure, Tertiary ,Substrate Specificity ,Archaeoglobus fulgidus ,Enzyme Stability ,Thermodynamics ,Amino Acid Oxidoreductases ,Amino Acid Sequence ,Protein Binding - Abstract
The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidus L-aspDH was refined at a resolution of 1.9 A with a crystallographic R-factor of 21.7% (R(free) = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidus L-aspDH/NAD/citrate ternary complex and the Thermotoga maritima L-aspDH/NAD binary complex showed that A. fulgidus L-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritima L-aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidus L-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two L-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to L-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic L-aspDH.
- Published
- 2007
50. The first crystal structure of an archaeal helical repeat protein
- Author
-
Nobuhiko Katunuma, Haruhiko Sakuraba, Kazunari Yoneda, Takeshi Kawabata, Hideaki Tsuge, Seiki Kuramitsu, and Toshihisa Ohshima
- Subjects
Models, Molecular ,Protein Folding ,Protein Conformation ,Archaeal Proteins ,Molecular Sequence Data ,Biophysics ,Sulfolobus tokodaii ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Structural genomics ,Sulfolobus ,Open Reading Frames ,Protein structure ,Tandem repeat ,Structural Biology ,Structural Genomics Communications ,Genetics ,Escherichia coli ,Amino Acid Sequence ,Cloning, Molecular ,Peptide sequence ,biology ,Sequence Homology, Amino Acid ,Superhelix ,Condensed Matter Physics ,biology.organism_classification ,Archaea ,Protein Structure, Tertiary ,Crystallography ,Protein folding ,Algorithms - Abstract
The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 A resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten alpha-helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein.
- Published
- 2005
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