title: A mutant form of 3-ketosteroid-Δ(1)-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by Mycobacterium neoaurum ST-095.
abstract: Mycobacterium neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD), produce very different molar ratios of ADD/AD. The distinct differences were related to the enzyme activity of 3-ketosteroid-Δ(1)-dehydrogenase (KSDD), which catalyzes the C1,2 dehydrogenation of AD to ADD specifically. In this study, by analyzing the primary structure of KSDDI (from M. neoaurum ST-095) and KSDDII (from M. neoaurum JC-12), we found the only difference between KSDDI and KSDDII was the mutation of Val(366) to Ser(366). This mutation directly affected KSDD enzyme activity, and this result was confirmed by heterologous expression of these two enzymes in Bacillus subtilis. Assay of the purified recombinant enzymes showed that KSDDII has a higher C1,2 dehydrogenation activity than KSDDI. The functional difference between KSDDI and KSDDII in phytosterol biotransformation was revealed by gene disruption and complementation. Phytosterol transformation results demonstrated that ksdd I and ksdd II gene disrupted strains showed similar ADD/AD molar ratios, while the ADD/AD molar ratios of the ksdd I and ksdd II complemented strains were restored to their original levels. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. Finally, KSDD structure analysis revealed that the Val(366)Ser mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD. This study provides a reliable theoretical basis for understanding the structure and catalytic mechanism of the Mycobacteria KSDD enzyme.
authors.affiliation: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Minglong
authors.initials: M
authors.lastname: Shao
authors.affiliation: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China. raozhm@jiangnan.edu.cn.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.affiliation: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.affiliation: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Taowei
authors.initials: T
authors.lastname: Yang
authors.affiliation: Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA.
authors.forename: Shangtian
authors.initials: S
authors.lastname: Yang
mesh_terms: D000730:Androstadienes / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D006865:Hydrogenation; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009161:Mycobacterium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D009170:Nontuberculous Mycobacteria / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010840:Phytosterols / Q000378:metabolism*
chemical_list: D000730:Androstadienes; D050505:Mutant Proteins; D010840:Phytosterols; C006573:1,4-androstadiene-3,17-dione; D000735:Androstenedione; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase
keywords: 3-Ketosteroid-Δ1-dehydrogenase; Mutation; Mycobacterium neoaurum; Phytosterol