Cloning, expression and characterization of a putative 2,5-diketo-d-gluconic acid reductase in Comamonas testosteroni

•We cloned a novel 2,5-diketo-d-gluconic acid reductase (2,5DKGR) gene from C. testosteroni ATCC11996.•The 2,5DKGR belongs to the aldo–keto reductases (AKRs) superfamily.•The 2,5DKGR expression could be induced by estradiol and methyltestosterone.•The growth and steroid degradation of 2,5DKGR knock-out mutant were impaired.•The 2,5DKGR may be involved in the metabolism of steroid compounds in C. testosteroni.

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.

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