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A mutant form of 3-ketosteroid-Δ1-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
- Shao, Minglong, Zhang, Xian, Rao, Zhiming, Xu, Meijuan, Yang, Taowei, Li, Hui, Xu, Zhenghong, Yang, Shangtian
- Journal of industrial microbiology & biotechnology 2016 v.43 no.5 pp. 691-701
- Bacillus subtilis, Mycobacterium neoaurum, biotransformation, dehydrogenation, enzyme activity, enzymes, gene targeting, genes, heterologous gene expression, mutants, mutation, phytosterols
- 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-Δ¹-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 Val³⁶⁶ to Ser³⁶⁶. 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 Val³⁶⁶Ser 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.