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dc.contributor.author | Takase, Ryuichi | en |
dc.contributor.author | Mikami, Bunzo | en |
dc.contributor.author | Kawai, Shigeyuki | en |
dc.contributor.author | Murata, Kousaku | en |
dc.contributor.author | Hashimoto, Wataru | en |
dc.contributor.alternative | 橋本, 渉 | ja |
dc.date.accessioned | 2016-01-18T04:54:43Z | - |
dc.date.available | 2016-01-18T04:54:43Z | - |
dc.date.issued | 2014-11-28 | - |
dc.identifier.issn | 0021-9258 | - |
dc.identifier.uri | http://hdl.handle.net/2433/203071 | - |
dc.description.abstract | The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharides to monosaccharides through four alginate lyase reactions. The resultant monosaccharide, which is nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), is further metabolized to 2-keto-3-deoxy-D-gluconate by NADPH-dependent reductase A1-R in the short-chain dehydrogenase/reductase (SDR) family. A1-R-deficient cells produced another DEH reductase, designated A1-R', with a preference for NADH. Here, we show the identification of a novel NADH-dependent DEH reductase A1-R' in strain A1, structural determination of A1-R' by x-ray crystallography, and structure-based conversion of a coenzyme requirement in SDR enzymes, A1-R and A1-R'. A1-R' was purified from strain A1 cells and enzymatically characterized. Except for the coenzyme requirement, there was no significant difference in enzyme characteristics between A1-R and A1-R'. Crystal structures of A1-R' and A1-R'·NAD(+) complex were determined at 1.8 and 2.7 Å resolutions, respectively. Because of a 64% sequence identity, overall structures of A1-R' and A1-R were similar, although a difference in the coenzyme-binding site (particularly the nucleoside ribose 2' region) was observed. Distinct from A1-R, A1-R' included a negatively charged, shallower binding site. These differences were caused by amino acid residues on the two loops around the site. The A1-R' mutant with the two A1-R-typed loops maintained potent enzyme activity with specificity for NADPH rather than NADH, demonstrating that the two loops determine the coenzyme requirement, and loop exchange is a promising method for conversion of coenzyme requirement in the SDR family. | en |
dc.format.mimetype | application/pdf | - |
dc.language.iso | eng | - |
dc.publisher | American Society for Biochemistry and Molecular Biology | en |
dc.rights | This research was originally published in [The Journal of Biological Chemistry, 289, 33198-33214. doi: 10.1074/jbc.M114.585661.] © the American Society for Biochemistry and Molecular Biology | en |
dc.rights | この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。 | ja |
dc.rights | This is not the published version. Please cite only the published version. | en |
dc.subject | Alginate Lyase | en |
dc.subject | Bacterial Metabolism | en |
dc.subject | Enzyme Kinetics | en |
dc.subject | Nicotinamide Adenine Dinucleotide (NADH) | en |
dc.subject | Reductase | en |
dc.subject | Site-directed Mutagenesis | en |
dc.subject | X-ray Crystallography | en |
dc.subject.mesh | Alginates/chemistry | en |
dc.subject.mesh | Alginates/metabolism | en |
dc.subject.mesh | Bacterial Proteins/chemistry | en |
dc.subject.mesh | Bacterial Proteins/metabolism | en |
dc.subject.mesh | Crystallography, X-Ray | en |
dc.subject.mesh | Glucuronic Acid/chemistry | en |
dc.subject.mesh | Glucuronic Acid/metabolism | en |
dc.subject.mesh | Hexuronic Acids/chemistry | en |
dc.subject.mesh | Hexuronic Acids/metabolism | en |
dc.subject.mesh | NADP/chemistry | en |
dc.subject.mesh | NADP/metabolism | en |
dc.subject.mesh | Oxidoreductases/chemistry | en |
dc.subject.mesh | Oxidoreductases/metabolism | en |
dc.subject.mesh | Protein Structure, Secondary | en |
dc.subject.mesh | Protein Structure, Tertiary | en |
dc.subject.mesh | Sphingomonas/enzymology | en |
dc.title | Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism. | en |
dc.type | journal article | - |
dc.type.niitype | Journal Article | - |
dc.identifier.ncid | AA00251083 | - |
dc.identifier.jtitle | The Journal of biological chemistry | en |
dc.identifier.volume | 289 | - |
dc.identifier.issue | 48 | - |
dc.identifier.spage | 33198 | - |
dc.identifier.epage | 33214 | - |
dc.relation.doi | 10.1074/jbc.M114.585661 | - |
dc.textversion | author | - |
dc.identifier.pmid | 25288804 | - |
dcterms.accessRights | open access | - |
出現コレクション: | 学術雑誌掲載論文等 |
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