title: Cloning and characterization of a novel β-ketoacyl-ACP reductase from Comamonas testosteroni.
abstract: Comamonas testosteroni (C. testosteroni) is a gram negative bacterium which can use steroid as a carbon source and degrade steroid with about 20 special enzymes. Most of the enzymes are inducible enzymes. 3-Oxoacyl-ACP reductase (E.C. 1.1.1.100) alternatively known as β-ketoacyl-ACP reductase (BKR) is involved in fatty acid syntheses. DNA sequence comparison showed that BKR belongs to the short-chain alcohol dehydrogenase (SDR) family. Our results showed that BKR is necessary for the degradation of steroid hormones in C. testosteroni. The DNA fragment of the BKR gene was cloned into an expressional plasmid pET-15b. BKR protein was expressed with 6× His-tag on the N-terminus and the enzyme was purified with Ni-column. Antibodies against BKR were prepared and a new BKR quantitative ELISA was created in our laboratory. The purified BKR is a 30.6 kDa protein on SDS-PAGE. C. testosteroni was induced by testosterone, estradiol, estriol and cholesterol. The expression of BKR was detected with an ELISA. The result showed that the BKR expression could be induced by cholesterol and estriol but not by testosterone and estradiol. BKR gene knock-out mutant (M-C.T.) was prepared by homologous integration. High performance liquid chromatography (HPLC) was used to detect steroid hormone degradation in C. testosteroni ATCC11996 and BKR gene knock-out mutant. We proved that the M-C.T. eliminated of testosterone degradation. Degradations of cholesterol and estradiol were also decreased. We conclude that the novel BKR in C. testosteroni plays an important role in steroid degradation. This work provides some new information of SDR and steroid degradation in C. testosteroni.
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: Ye
authors.initials: Y
authors.lastname: Ji
authors.affiliation: School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
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: D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase / Q000235:genetics*; D000426:Alcohol Dehydrogenase / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002784:Cholesterol / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004958:Estradiol / Q000235:genetics; D004964:Estriol / Q000235:genetics; D064429:Fatty Acid Synthases / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016415:Sequence Alignment; D013256:Steroids / Q000378:metabolism; D013739:Testosterone / Q000235:genetics
chemical_list: D013256:Steroids; D013739:Testosterone; D004958:Estradiol; D002784:Cholesterol; D000426:Alcohol Dehydrogenase; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; C036543:short chain trans-2-enoyl-CoA reductase; D009247:NADH, NADPH Oxidoreductases; D064429:Fatty Acid Synthases; D004964:Estriol
keywords: BKR; Comamonas testosteroni; Gene knock-out; SDR; Short-chain dehydrogenase/reductase; β-Ketoacyl-ACP reductase