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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 ![]() ![]() ![]() Mikami, Bunzo ![]() ![]() Kawai, Shigeyuki ![]() ![]() Murata, Kousaku Hashimoto, Wataru ![]() ![]() ![]() |
Author's alias: | 橋本, 渉 |
Keywords: | Alginate Lyase Bacterial Metabolism Enzyme Kinetics Nicotinamide Adenine Dinucleotide (NADH) Reductase 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。 |
URI: | http://hdl.handle.net/2433/203071 |
DOI(Published Version): | 10.1074/jbc.M114.585661 |
PubMed ID: | 25288804 |
Appears in Collections: | Journal Articles |

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