Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism [Protein Structure and Folding]

October 6th, 2014 by Takase, R., Mikami, B., Kawai, S., Murata, K., Hashimoto, W.

The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharide to monosaccharides through four alginate lyases 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, structure 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 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 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.
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