4TKM

Crystal structure of NADH-dependent reductase A1-R' complexed with NAD

Summary for 4TKM

• Entry DOI: 10.2210/pdb4tkm/pdb
• Related: 4TKL
• Descriptor: NADH-dependent reductase for 4-deoxy-L-erythro-5-hexoseulose uronate, SULFATE ION, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, ... (4 entities in total)
• Functional Keywords: alpha/beta/alpha, rossmann-fold, alginate metabolism, short-chain dehydrogenase/reductase, oxidoreductase
• Biological source: Sphingomonas sp. A1
• Total number of polymer chains: 2
• Total formula weight: 55881.90

Authors

Takase, R.,Mikami, B.,Kawai, S.,Murata, K.,Hashimoto, W. (deposition date: 2014-05-27, release date: 2014-06-25, Last modification date: 2023-11-08)

Primary citation

Takase, R.,Mikami, B.,Kawai, S.,Murata, K.,Hashimoto, W.
Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism.
J.Biol.Chem., 289:33198-33214, 2014

PubMed 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.

PubMed: 25288804
DOI: 10.1074/jbc.M114.585661

Experimental method

X-RAY DIFFRACTION (2.67 Å)