title: Cloning, expression and characterization of a putative 2,5-diketo-D-gluconic acid reductase in Comamonas testosteroni.
abstract: Aldo-keto reductases (AKRs) are a superfamily of soluble NAD(P)(H) oxidoreductases. The function of the enzymes is to reduce aldehydes and ketones into primary and secondary alcohols. We have cloned a 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene from Comamonas testosteroni (C. testosteroni) ATCC11996 (a Gram-negative bacterium which can use steroids as carbon and energy source) into plasmid pET-15b and over expressed in Escherichia coli BL21 (DE3). The protein was purified by His-tag Metal chelating affinity chromatography column. The 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene contains 1062 bp and could be translated into a protein of 353 amino acid residues. Three consensus sequences of the AKR superfamily are found as GxxxxDxAxxY, LxxxGxxxPxxGxG and LxxxxxxxxxDxxxxH. GxxxxDxAxxY is the active site, LxxxGxxxPxxGxG is the Cofactor-binding site for NAD(P)(H), LxxxxxxxxxDxxxxH is used for supporting the 3D structure. 2,5-diketo-D-gluconic acid reductase gene of C. testosteroni was knocked out and a mutant M-AKR was obtained. Compared to wild type C. testosteroni, degradations of testosterone, estradiol, oestrone and methyltestosterone in mutant M-AKR were decreased. Therefore, 2,5-diketo-D-gluconic acid reductase in C. testosteroni is involved in steroid degradation.
authors.affiliation: School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, PR China.
authors.forename: Yuanan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ji
authors.affiliation: School of Science, Changchun University of Science and Technology, Changchun 130022, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, PR China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, PR China. Electronic address: yuyuanhua8888@126.com.
authors.forename: Yuanhua
authors.initials: Y
authors.lastname: Yu
mesh_terms: D000449:Aldehyde Reductase / Q000235:genetics*; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000235:genetics; D009249:NADP / Q000235:genetics; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D016415:Sequence Alignment; D013256:Steroids / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics*
chemical_list: D013256:Steroids; D009243:NAD; D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
keywords: 2,5-Diketo-d-gluconic acid reductase; Aldo–keto reductase; Comamonas testosteroni; Knock out mutant