TY - JOUR T1 - Oxygen surrogate systems for supporting human drug-metabolizing cytochrome P450 enzymes JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.120.090555 SP - dmd.120.090555 AU - Silja J Strohmaier AU - James J De Voss AU - Ulrik Jurva AU - Shalini Andersson AU - Elizabeth M. J. Gillam Y1 - 2020/01/01 UR - http://dmd.aspetjournals.org/content/early/2020/03/31/dmd.120.090555.abstract N2 - Oxygen surrogates (OSs) have been used to support cytochrome P450 enzymes for diverse purposes in drug metabolism research, including reaction phenotyping, mechanistic and inhibition studies, studies of redox partner interactions, and to avoid the need for NADPH or a redox partner. They also have been used in engineering P450s for more cost-effective, NADPH-independent biocatalysis. However, despite their broad application little is known of the preference of individual P450s for different OSs, or the substrate dependence of OS-supported activity. Furthermore, the biocatalytic potential of OSs other than cumene hydroperoxide (CuOOH) and hydrogen peroxide (H2O2) is yet to be explored. Here, we investigated the ability of the major human drug-metabolizing P450s, namely CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP1A2, to use different OSs: H2O2; tert-butyl hydroperoxide (tert-BuOOH); CuOOH; (diacetoxyiodo)benzene (BAIB); and bis(trifluoroacetoxy)iodobenzene (F-BAIB). Overall, CuOOH and tert-BuOOH were found to be the most effective at supporting these P450s. However, the ability of P450s to be supported by OSs effectively was also found to be highly dependent on the substrate used. This suggests that the choice of OS should be tailored to both the P450 and the substrate under investigation, underscoring the need to employ screening methods in directed evolution experiments that reflect the activity towards the substrate of interest to the end application.SIGNIFICANCE STATEMENT Cytochrome P450 enzymes can be supported by different oxygen surrogates (OSs), avoiding the need for a redox partner and costly NADPH. However few data exist comparing relative activity with different OSs and substrates. This study shows that the choice of OS used to support the major drug-metabolizing P450s influences their relative activity and regioselectivity in a substrate specific fashion, and provides a model for the more efficient use of P450s for metabolite biosynthesis. ER -