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Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2014 Nov 28; Vol. 289 (48), pp. 33198-214. Date of Electronic Publication: 2014 Oct 06. - Publication Year :
- 2014
-
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.<br /> (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Alginates metabolism
Bacterial Proteins metabolism
Crystallography, X-Ray
Glucuronic Acid chemistry
Glucuronic Acid metabolism
Hexuronic Acids chemistry
Hexuronic Acids metabolism
NADP metabolism
Oxidoreductases metabolism
Protein Structure, Secondary
Protein Structure, Tertiary
Alginates chemistry
Bacterial Proteins chemistry
NADP chemistry
Oxidoreductases chemistry
Sphingomonas enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 289
- Issue :
- 48
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 25288804
- Full Text :
- https://doi.org/10.1074/jbc.M114.585661