論文

査読有り
2014年11月

Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism

JOURNAL OF BIOLOGICAL CHEMISTRY
  • Ryuichi Takase
  • ,
  • Bunzo Mikami
  • ,
  • Shigeyuki Kawai
  • ,
  • Kousaku Murata
  • ,
  • Wataru Hashimoto

289
48
開始ページ
33198
終了ページ
33214
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1074/jbc.M114.585661
出版者・発行元
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

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 inSDRenzymes, 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 angstrom 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.

リンク情報
DOI
https://doi.org/10.1074/jbc.M114.585661
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/25288804
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000345636600014&DestApp=WOS_CPL
ID情報
  • DOI : 10.1074/jbc.M114.585661
  • ISSN : 0021-9258
  • eISSN : 1083-351X
  • PubMed ID : 25288804
  • Web of Science ID : WOS:000345636600014

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