Journal of Microbiology and Biotechnology

  • 제30권11호
  • /
  • Pages.1750-1759
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  • 2020
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952

  • Rimal, Hemraj (Department of Life Science and Biochemical Engineering, Graduate School, SunMoon University) ;
  • Subedi, Pradeep (Department of Life Science and Biochemical Engineering, Graduate School, SunMoon University) ;
  • Kim, Ki -Hwa (Department of Life Science and Biochemical Engineering, Graduate School, SunMoon University) ;
  • Park, Hyun (Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Lee, Jun Hyuck (Unit of Research for Practical Application, Korea Polar Research Institute) ;
  • Oh, Tae-Jin (Department of Life Science and Biochemical Engineering, Graduate School, SunMoon University)
  • 투고 : 2020.07.02
  • 심사 : 2020.09.11
  • 발행 : 2020.11.28
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초록

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated KD value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min-1 nmol-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

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