Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Crystal Structure and Biochemical Analysis of a Cytochrome P450 Steroid Hydroxylase (BaCYP106A6) from Bacillus Species

  • Ki-Hwa Kim (Department of Life Science and Biochemical Engineering, Sunmoon University) ;
  • Hackwon Do (Research Unit of Cryogenic Novel Materials, Korea Polar Research Institute) ;
  • Chang Woo Lee (Research Unit of Cryogenic Novel Materials, Korea Polar Research Institute) ;
  • Pradeep Subedi (Department of Life Science and Biochemical Engineering, Sunmoon University) ;
  • Mieyoung Choi (Department of Pharmaceutical Engineering and Biotechnology, Sunmoon University) ;
  • Yewon Nam (Research Unit of Cryogenic Novel Materials, Korea Polar Research Institute) ;
  • Jun Hyuck Lee (Research Unit of Cryogenic Novel Materials, Korea Polar Research Institute) ;
  • Tae-Jin Oh (Department of Life Science and Biochemical Engineering, Sunmoon University)
  • Received : 2022.11.16
  • Accepted : 2022.12.06
  • Published : 2023.03.28
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Abstract

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

Keywords

Acknowledgement

This research was part of the project titled "Development of potential antibiotic compounds using polar organism resources (20200610, KOPRI Grant PM23030)," funded by the Ministry of Oceans and Fisheries, Korea. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2019R1D1A3A03103903). We thank the Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chungbuk, Korea, for NMR analyses.

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