Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Crystal Structure and Functional Characterization of a Cytochrome P450 (BaCYP106A2) from Bacillus sp. PAMC 23377

  • Kim, Ki-Hwa (Department of Life Science and Biochemical Engineering, Sunmoon University) ;
  • Lee, Chang Woo (Unit of Polar Genomics, Korea Polar Research Institute) ;
  • Dangi, Bikash (Department of Life Science and Biochemical Engineering, Sunmoon University) ;
  • Park, Sun-Ha (Unit of Polar Genomics, Korea Polar Research Institute) ;
  • Park, Hyun (Unit of Polar Genomics, Korea Polar Research Institute) ;
  • Oh, Tae-Jin (Department of Life Science and Biochemical Engineering, Sunmoon University) ;
  • Lee, Jun Hyuck (Unit of Polar Genomics, Korea Polar Research Institute)
  • Received : 2017.06.07
  • Accepted : 2017.06.19
  • Published : 2017.08.28
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Abstract

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity ($K_d$ value) to 4-androstenedione was $387{\pm}37{\mu}M$. Moreover, the crystal structure of BaCYP106A2 was determined at $2.7{\AA}$ resolution. Structural analysis suggested that the ${\alpha}8-{\alpha}9$ loop region of BaCYP106A2 is intrinsically mobile and might be important for initial ligand binding. The hydroxyl activity of BaCYP106A2 was identified using in vitro enzyme assays. Its activity was confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using chromatography and mass spectrometry methods. The main products were mono-hydroxylated compounds with high conversion yields. This is the second study on the structure of CYP106A steroid hydroxylases, and should contribute new insight into the interactions of bacterial CYP106A with steroid substrates, providing baseline data for studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

Keywords

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