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Publication Abstract from PubMed

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 A 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.

Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism.,Takase R, Mikami B, Kawai S, Murata K, Hashimoto W J Biol Chem. 2014 Nov 28;289(48):33198-214. doi: 10.1074/jbc.M114.585661. Epub, 2014 Oct 6. PMID:25288804^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.