Site-directed mutagenesis under the direction of in silico protein docking modeling reveals the active site residues of 3-ketosteroid-Δ 1 -dehydrogenase from Mycobacterium neoaurum

• Published in: World journal of microbiology & biotechnology 2017-07, Vol.33 (7), p.146
• Main Authors: Qin, Ning, Shen, Yanbing, Yang, Xu, Su, Liqiu, Tang, Rui, Li, Wei, Wang, Min
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Catalytic Domain; Computer Simulation; Crystallography, X-Ray; Models, Molecular; Molecular Docking Simulation; Mutagenesis, Site-Directed; Mycobacterium - enzymology; Mycobacterium - genetics; Oxidoreductases - chemistry; Oxidoreductases - genetics
• ISSN: 1573-0972

3-Ketosteroid-Δ -dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.