Characterisation of a Self-Sufficient Cytochrome P450 Enzyme From the Bacterium Thermosporothrix hazakensis and Its Conversion Into a Peroxygenase

Abstract

The cytochrome P450 monooxygenase enzymes (CYPs) of the CYP102 family are versatile, self-sufficient biocatalysts. The archetypal example is CYP102A1 (P450_BM3) from the bacterium Bacillus (Priestia) megaterium, and variants of this enzyme can oxidise many substrates with high activity and selectivity. However, this enzyme has relatively low thermal stability. Here, we identify and characterise a CYP102 family enzyme from the moderately thermophilic bacterium Thermosporothrix hazakensis. We were able to produce this enzyme using Escherichia coli and demonstrate the in vivo oxidation of fatty acids. However, the activity of the isolated holoenzyme was low, so we generated a peroxygenase variant by introducing the E278Q and T279E mutations into the heme domain (‘HazakQE’). This isolated variant was able to catalyse the oxidation of a range of substrates using hydrogen peroxide as the oxidant. The product distributions arising from fatty acid oxidation using the holoprotein monooxygenase and heme domain peroxygenase variants of this enzyme were broadly similar to those obtained with P450_BM3. For fatty acids, the oxidation occurred predominantly at the ω-1 through to ω-3 positions. Styrene was epoxidised and tetralone hydroxylated at the benzylic carbon. The oxidation of 1-methoxynaphthalene generated the dimeric Russig's blue, enabling colorimetric assays of the enzyme activity. Although the HazakQE heme peroxygenase was more thermostable than the mesophilic CYP199A4 enzyme from Rhodopseudomonas palustris, it was not more resistant to heating than the heme domain of P450_BM3. These peroxygenase variants offer a simple platform for metabolite identification and biocatalysts for oxidation reactions, which could be enhanced through protein engineering.