TY - JOUR
T1 - An integrated screening system for the selection of exemplary substrates for natural and engineered cytochrome P450s
AU - Kanoh, Naoki
AU - Kawamata-Asano, Ayano
AU - Suzuki, Kana
AU - Takahashi, Yusuke
AU - Miyazawa, Takeshi
AU - Nakamura, Takemichi
AU - Moriya, Takashi
AU - Hirano, Hiroyuki
AU - Osada, Hiroyuki
AU - Iwabuchi, Yoshiharu
AU - Takahashi, Shunji
N1 - Funding Information:
We thank Prof. Shingo Nagano (Tottori University), Prof. Stephen G. Sligar (University of Illinois), Prof. Yoshihito Watanabe and Prof. Osami Shoji (Nagoya University) for the generous gift of DNA constructs, Prof. Manfred T. Reetz (Max Planck Institute for Coal Research) for the generous gift of DNA constructs and for the critical reading of and comments on the early version of this manuscript. This work was supported by a Grant-in-Aid for Scientific Research on the Innovative Area “Chemical Biology of Natural Products” from The Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 23102013 to NK), by the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Japan Agency for Medical Research and Development (AMED) (No. JP18am0101100), by Research Fellowship Grants for Young Researchers from the Japan Society for the Promotion of Science (No. 15J03635 to AK, and No. 257508 to TM), and by the Naito Foundation.
Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of “exemplary” substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acquired an ability to hydroxylate non-natural substrate steroids regio- and stereoselectively by two amino acid mutations. The distinct transition of exemplary substrates due to each single amino acid mutation was revealed, demonstrating the utility of the established system.
AB - Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of “exemplary” substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acquired an ability to hydroxylate non-natural substrate steroids regio- and stereoselectively by two amino acid mutations. The distinct transition of exemplary substrates due to each single amino acid mutation was revealed, demonstrating the utility of the established system.
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U2 - 10.1038/s41598-019-54473-8
DO - 10.1038/s41598-019-54473-8
M3 - Article
C2 - 31792277
AN - SCOPUS:85075917858
SN - 2045-2322
VL - 9
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 18023
ER -