Efficient isoprenoid oxidation by an engineered heme peroxygenase from the magnetotactic bacterium Magnetospirillum gryphiswaldense

• A new cytochrome P450 enzyme from Magnetospirillum gryphiswaldense , was characterised. • The enzyme binds a range of isoprenoids including bisabolol, β-ionone and steroids. • Protein engineering was used to convert the enzyme to a peroxygenase enzyme. • The enzyme catalyzed the selective hydroxylation of progesterone and β-ionone. • The oxidation of bisabolol generated two isomers of 5‑hydroxy-α-bisabolol. Cytochrome P450 enzymes (CYPs) are natural monooxygenases, which can catalyze the selective oxidation of organic compounds, using dioxygen. The newly discovered CYP1597A2, the only CYP in the magnetotactic bacterium, Magnetospirillum gryphiswaldense , was demonstrated to bind a range of isoprenoids including bisabolol, β-ionone, progesterone, and testosterone with bisabolol and progesterone having the best fit with the active site or the highest affinity. This enzyme was converted into a more efficient peroxygenase, by mutation of residues in the I-helix, giving it the ability to oxidize these substrates using H 2 O 2 . It catalyzed the hydroxylation of progesterone into single major product, 2-hydroxyprogesterone. The oxidation of testosterone was less selective, generating 15- and 16α-hydroxytestosterone and androstenedione as the three major metabolites. The hydroxylation of β-ionone into ( S )-4‑hydroxy-β-ionone was stereoselective (97 % e.e.). The oxidation of bisabolol generated several metabolites, with (1 R ,5 S ,7 S )-5‑hydroxy-α-bisabolol, along with its (1 R ,5 R ,7 S ) isomer, being the major products. In this study, we demonstrated that CYP1597A2 from M. gryphiswaldense , has a versatile substrate range, so it could provide a platform for the modification of active site residues, through protein engineering, to generate bespoke and efficient isoprenoid oxidizing biocatalysts.