Functional characterization of a novel CYP119 variant to explore its biocatalytic potential

Abstract

Biocatalysts are increasingly applied in the pharmaceutical and chemical industry. Cytochrome P450 enzymes (P450s) are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. P450s catalyze reactions using nicotinamide adenine dinucleotide phosphate (NAD(P)H) cofactor and electron transfer proteins. Alternatively, P450s can utilize hydrogen peroxide (H 2 O 2 ) as an oxidant, but this pathway is inefficient. P450s that show higher efficiency with peroxides are sought after in industrial applications. P450s from thermophilic organisms have more potential applications as they are stable toward high temperature, high and low pH, and organic solvents. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius. In our previous study, a novel T213R/T214I (double mutant [DM]) variant of CYP119 was obtained by screening a mutant library for higher peroxidation activity utilizing H 2 O 2 . Here, we characterized the substrate scope; stability toward peroxides; and temperature and organic solvent tolerance of DM CYP119 to identify its potential as an industrial biocatalyst. DM CYP119 displayed higher stability than wild-type (WT) CYP119 toward organic peroxides. It shows higher peroxidation activity for nonnatural substrates and higher affinity for progesterone and other bioactive potential substrates compared to WT CYP119. DM CYP119 emerges as a new biocatalyst with a wide range of potential applications in the pharmaceutical and chemical industry.

Microbial P450 repertoire (P450ome) and its application feasibility in pharmaceutical industry, chemical industry, and environmental protection

“…CYP109E1 (Jóźwik et al, 2016) is able to hydroxylate testosterone at position 16β. The CYP119 variant T213R/T214I shows higher peroxidation activity for nonnatural substrates and higher affinity for progesterone and other bioactive potential substrates (Başlar et al, 2020; Sakalli & Surmeli, 2021). From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450‐designated as P450‐T3, which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected and can bound and converted testosterone (Nguyen et al, 2020a).…”

“…CYP109E1 (Jóźwik et al, 2016) is able to hydroxylate testosterone at position 16β. The CYP119 variant T213R/T214I shows higher peroxidation activity for nonnatural substrates and higher affinity for progesterone and other bioactive potential substrates (Başlar et al, 2020;Sakalli & Surmeli, 2021). From the metagenomics library of the Binh Chau hot…”

Evolution of Cytochrome P450 Enzymes and Their Redox Partners in Archaea

“…Information on the evolution of P450s in bacteria, eukarya and viruses is available, but not in archaea. Archaea have P450s and one of the P450s, CYP119 from Sulfolobus solfataricus , has been subjected to intense investigation due to its thermophilic properties [ 23 , 24 , 25 , 26 , 27 , 28 ].…”