Speaker
Description
Background: Drug metabolism studies play a critical role in drug development by enabling the prediction of pharmacokinetics and potential drug–drug interactions. Conventional cytochrome P450 enzymes are commonly used for these studies. Unspecific peroxygenases (UPOs), heme-thiolate enzymes structurally related to P450s, have recently emerged as promising alternatives. Unlike P450s, UPOs utilize inexpensive hydrogen peroxide (H₂O₂) as an oxidant and do not require redox partners and NAD(P)H. These properties make them attractive for cost-effective, scalable production of drug metabolites. In this study, we aimed to evaluate the catalytic potential of a UPO from Myceliophthora thermophila (MthUPO) and to assess its applicability for metabolite production of drugs.
Methods: MthUPO gene, derived from fungi, was cloned into the pET28a expression vector. The plasmids were transformed into Escherichia coli C2566 and the recombinant cells were cultured in Terrific Broth. The soluble protein was purified by Ni-NTA affinity chromatography. Ibuprofen, diclofenac, and fluvastatin served as substrates for the enzyme reaction. The reaction products were analyzed by HPLC.
Results: MthUPO catalyzed the hydroxylation of fluvastatin, ibuprofen, and diclofenac. Ibuprofen yielded three hydroxylated metabolites and carboxyibuprofen. Fluvastatin produced two hydroxylated major metabolites and one minor metabolite. Diclofenac yielded one hydroxylated metabolite. MthUPO exhibited high conversion rates of ibuprofen and fluvastatin reaching up to 30%.
Conclusions: We investigated the enzymatic hydroxylation activity of MthUPO toward drugs. MthUPO efficiently catalyzes the oxidation of ibuprofen, diclofenac, and fluvastatin which are primarily metabolized by CYP2C9 in vivo. These results suggest that MthUPO is capable of catalyzing the oxidation of relevant CYP2C9 substrates and may serve as a promising biocatalyst candidate for drug metabolism studies and metabolite production.
