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

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.

SUBMITTER: Kanoh N

PROVIDER: S-EPMC6888865 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Kanoh Naoki N Kawamata-Asano Ayano A Suzuki Kana K Takahashi Yusuke Y Miyazawa Takeshi T Nakamura Takemichi T Moriya Takashi T Hirano Hiroyuki H Osada Hiroyuki H Iwabuchi Yoshiharu Y Takahashi Shunji S

Scientific reports 20191202 1

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" ...[more]

PMID: 31792277

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Project description:The superfamily of cytochrome P450s forms a large class of heme monooxygenases with more than 13,000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochrome P450s from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochrome P450s reveals the presence of two distinct structural classes of cytochrome P450s. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which may result in an increase of the volume of the substrate binding pocket and an opening of a potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochrome P450s, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochrome P450s can be rationalized through the presence of several amino acid inserts in the latter class which are involved in direct interactions with the membrane, namely the F'- and G'-helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B-C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B-C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochrome P450s with important functional implications induced by the lipid bilayer.

Dataset Information

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

Publications

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

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