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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