An integrated screening system for the selection of exemplary substrates for natural and engineered cytochrome P450s

Naoki Kanoh,Hiroyuki Hirano,Takashi Moriya,Kana Suzuki,Takemichi Nakamura,Takeshi Miyazawa,Yusuke Takahashi,Ayano Kawamata-Asano,Yoshiharu Iwabuchi,Shunji Takahashi,Hiroyuki Osada

doi:10.1038/s41598-019-54473-8

Naoki Kanoh, Hiroyuki Hirano + Show 9 more

Open Access

https://doi.org/10.1038/s41598-019-54473-8

Copy DOI

Abstract

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.

Highlights

Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases
Cytochrome P450s (P450s) constitute an exceptional superfamily of monooxygenases found in living organisms from microorganisms to plants to humans, and are responsible for drug metabolism and biosynthesis of secondary metabolites[1]
Designing the function of P450s would be possible if sufficient data on the influence of amino acid mutations in the substrate-binding pocket on substrate selectivity were accumulated for the P450 of interest

Summary

Introduction

Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. 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. High activity of P450 BM3 and its good functional expression in Escherichia coli have www.nature.com/scientificreports made it an attractive platform for engineering aimed at the selective oxidation of unnatural substrates and the production of useful compounds[17]. By combining the four established methods, we constructed an integrated P450 substrate screening system for the selection of exemplary substrates that (1) are typically recognized by the P450 enzyme, (2) induce type I spectral change, (3) are rapidly oxidized in a highly coupled manner, and (4) are converted to a limited number of their oxidized products. Comparison of exemplary substrates for P450 BM3 wild-type (WT), P450 BM3 (F87A), and P450 BM3 (F87A/A330W) was performed, demonstrating a distinct transition of exemplary substrates due to each single amino acid mutation

Methods

Results

Conclusion