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Development of an improved Amplex Red peroxidation activity assay for screening cytochrome P450 variants and identification of a novel mutant of the thermophilic CYP119

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Abstract

Biocatalysts are increasingly utilized in the synthesis of drugs and agrochemicals as an alternative to chemical catalysis. They are preferred in the synthesis of enantiopure products due to their high regioselectivity and enantioselectivity. Cytochrome P450 (P450) oxygenases are valuable biocatalysts, since they catalyze the oxidation of carbon–hydrogen bonds with high efficiency and selectivity. However, practical use of P450s is limited due to their need for expensive cofactors and electron transport partners. P450s can employ hydrogen peroxide (H2O2) as an oxygen and electron donor, but the reaction with H2O2 is inefficient. The development of P450s that can use H2O2 will expand their applications. Here, an assay that utilizes Amplex Red peroxidation, to rapidly screen H2O2-dependent activity of P450 mutants in cell lysate was developed. This assay was employed to identify mutants of CYP119, a thermophilic P450 from Sulfolobus acidocaldarius, with increased peroxidation activity. A mutant library of CYP119 containing substitutions in the heme active site was constructed via combinatorial active-site saturation test and screened for improved activity. Screening of 158 colonies led to five mutants with higher activity. Among improved variants, T213R/T214I was characterized. T213R/T214I exhibited fivefold higher kcat for Amplex Red peroxidation and twofold higher kcat for styrene epoxidation. T213R/T214I showed higher stability towards heme degradation by H2O2. While the Km for H2O2 and styrene were not altered by the mutation, a fourfold decrease in the affinity for another substrate, lauric acid, was observed. In conclusion, Amplex Red peroxidation screening of CYP119 mutants yielded enzymes with increased peroxide-dependent activity.

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Abbreviations

P450:

Cytochrome P450

HRP:

Horseradish peroxidase

WT:

Wild type

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

NAD(P)H:

Nicotinamide adenine dinucleotide phosphate

CAST:

Combinatorial active-site saturation test

PMSF:

Phenylmethylsulfonyl fluoride

EDTA:

Ethylenediaminetetraacetic acid

DMSO:

Dimethyl sulfoxide

TBHP:

Tert-butyl hydroperoxide

FAD:

Flavin adenine dinucleotide

FMN:

Flavin mononucleotide

RCSB PDB:

The Research Collaboratory for Structural Bioinformatics Protein Data Bank

PDB ID:

Protein data bank identity

REU:

Rosetta energy unit

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Acknowledgements

This work was supported by The Scientific and Technological Research Council of Turkey [TUBİTAK, 116Z380]. We thank Ali Oğuz Büyükkileci for his help with the HPLC analysis and Çağlar Karakaya for allowing access to ultrasonicator instrument.

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Authors and Affiliations

  1. Department of Bioengineering, İzmir Institute of Technology, Gülbahçe, Urla, Izmir, Turkey

    M. Semih Başlar, Gülce Güralp, Ekin Kestevur Doğru & Nur Basak Surmeli

  2. Program in Biotechnology and Bioengineering, İzmir Institute of Technology, Gülbahce, Urla, Izmir, Turkey

    Tuğçe Sakallı & Emre Haklı

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  1. M. Semih Başlar
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  2. Tuğçe Sakallı
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  3. Gülce Güralp
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Correspondence to Nur Basak Surmeli.

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Başlar, M.S., Sakallı, T., Güralp, G. et al. Development of an improved Amplex Red peroxidation activity assay for screening cytochrome P450 variants and identification of a novel mutant of the thermophilic CYP119. J Biol Inorg Chem 25, 949–962 (2020). https://doi.org/10.1007/s00775-020-01816-w

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  • DOI: https://doi.org/10.1007/s00775-020-01816-w

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