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Title: Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism.
Authors: Takase, Ryuichi  kyouindb  KAKEN_id  orcid (unconfirmed)
Mikami, Bunzo  kyouindb  KAKEN_id
Kawai, Shigeyuki  kyouindb  KAKEN_id
Murata, Kousaku
Hashimoto, Wataru  kyouindb  KAKEN_id  orcid (unconfirmed)
Author's alias: 橋本, 渉
Keywords: Alginate Lyase
Bacterial Metabolism
Enzyme Kinetics
Nicotinamide Adenine Dinucleotide (NADH)
Site-directed Mutagenesis
X-ray Crystallography
Issue Date: 28-Nov-2014
Publisher: American Society for Biochemistry and Molecular Biology
Journal title: The Journal of biological chemistry
Volume: 289
Issue: 48
Start page: 33198
End page: 33214
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.
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
This is not the published version. Please cite only the published version. 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。
DOI(Published Version): 10.1074/jbc.M114.585661
PubMed ID: 25288804
Appears in Collections:Journal Articles

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