Genetic differences in ksdD influence on the ADD/AD ratio of Mycobacterium neoaurum

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• 作者：Rili Xie ; Yanbing Shen ; Ning Qin ; Yibo Wang…
• 关键词：Mycobacterium neoaurum ; 3 ; ketosteroid ; Δ1 ; dehydrogenase ; Biotransformation ; Androst ; 4 ; ene ; 3 ; 17 ; dione ; Androst ; 1 ; 4 ; diene ; 3 ; 17 ; dione
• 刊名：Journal of Industrial Microbiology and Biotechnology
• 出版年：2015
• 出版时间：April 2015
• 年：2015
• 卷：42
• 期：4
• 页码：507-513
• 全文大小：557 KB
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  10. Knol, J, Bodewits, K, Hessels, GI, Dijkhuizen, L, Geize, R (2008) 3-Keto-5α-steroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism. Biochem J 410: pp. 339-346 CrossRef
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• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Chemistry
  Biotechnology
  Genetic Engineering
  Biochemistry
  Bioinformatics
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1476-5535

文摘

Mycobacterium neoaurum TCCC 11028 (MNR) and M. neoaurum TCCC 11028 M3 (MNR M3) significantly differ in the ratio of androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) produced. The large fluctuations are related to the dehydrogenation activity of 3-ketosteroid-Δ1-dehydrogenase (KsdD). Analysis of the primary structure of KsdD showed that the Ser-138 of KsdD-MNR changed to Leu-138 of KsdD-MNR M3 because of C413T in the ksdD gene. This phenomenon directly affected KsdD activity. The effect of the primary structure of KsdD on dehydrogenation activity was confirmed through exogenous expression. Whole-cell transformation initially revealed that KsdD-MNR showed a higher dehydrogenation activity than KsdD-MNR M3. Then, ksdD gene replacement strain was constructed by homologous recombination. The results of steroid transformation experiments showed that the ability of the MNR M3ΔksdD::ksdD-MNR strain to produce ADD was improved and it returned to the similar level of the MNR strain. This result indicated that the ADD/AD ratio of the two M. neoaurum strains was influenced by the difference in ksdD. The mechanism by which residue mutations alter enzyme activity may be connected with the crystal structure of KsdD from Rhodococcus erythropolis SQ1. As a key amino acid residue in the active center position, Ser-138 played an important role in maintaining the active center in the hydrophobic environment of KsdD. This study may serve as a basis for future studies on the structural analysis and catalytic mechanism of dehydrogenase.