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.
Subject(s)
Aldehyde Reductase/genetics , Comamonas testosteroni/enzymology , Comamonas testosteroni/genetics , Sugar Alcohol Dehydrogenases/genetics , Aldo-Keto Reductases , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular/methods , Escherichia coli/enzymology , Escherichia coli/genetics , Molecular Sequence Data , Mutation/genetics , NAD/genetics , NADP/genetics , Phylogeny , Plasmids/genetics , Sequence Alignment , Steroids/metabolismABSTRACT
The short-chain dehydrogenase/reductase (SDR) superfamily is a large and diverse group of genes with members found in all forms of life. Comamonas testosteroni (C. testosterone) ATCC11996 is a Gram-negative bacterium which can use steroids as carbon and energy source. In the present investigation, we found a novel SDR gene 7alpha-hydroxysteroid dehydrogenase (7α-HSD) which is located 11.9 kb upstream from hsdA with the same transcription orientation in the C. testosteroni genome. The open reading frame of this putative 7alpha-hydroxysteroid dehydrogenase gene consists of 771 bp and translates into a protein of 256 amino acids. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 161-165 in the 7α-HSD sequence) essential for catalytic activity of SDR proteins. To produce purified 7α-HSD protein, the 7α-HSD gene was cloned into plasmid p(ET-15b) and the over expressed protein was purified by His-tag sequence on metal chelate chromatography. To prove that 7α-HSD is involved in the metabolic pathway of steroid compounds, we constructed a 7α-HSD knock-out mutant of C. testosteroni. Compared to the wild type C. testosteroni, degradation of testosterone, estradiol and cholesterol were decreased in the 7α-HSD knock-out mutant. Furthermore, growth in the medium with testosterone, estradiol and cholesterol was impaired in 7α-HSD knock-out mutant. The results showed that 7α-HSD is involved in steroid degradation.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cloning, Molecular , Comamonas testosteroni/enzymology , Hydroxysteroid Dehydrogenases/chemistry , Hydroxysteroid Dehydrogenases/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Comamonas testosteroni/chemistry , Comamonas testosteroni/genetics , Gene Expression Regulation, Bacterial , Hydroxysteroid Dehydrogenases/metabolism , Molecular Sequence Data , Sequence AlignmentABSTRACT
We expressed 17-hydroxysteroid dehydrogenase10 (17ß-hsd10) recombinant protein, prepared anti-17ß- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17ß-hsd10. RT-PCR was used to get the gene of 17ß-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17ß-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-ß-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17ß-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17ß-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17ß-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.
