Uncoupled human flavin-containing monooxygenase 3 releases superoxide radical in addition to hydrogen peroxide.

Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolizing enzyme capable of performing N- or S-oxidation using the C4a-hydroperoxy intermediate. In this work, we employ both wild type hFMO3 as well as an active site polymorphic variant (N61S) to unravel the uncoupling reactions in the catalytic cycle of this enzyme. We demonstrate that in addition to H2O2 this enzyme also produces superoxide anion radicals as its uncoupling products. The level of uncoupling was found to vary between 50 and 70% (WT) and 90-98% (N61S) for incubations with NADPH and benzydamine over a period of 5 or 20 min, respectively. For the first time, we were able to follow the production of the superoxide radical in hFMO3, which was found to account for 13-18% of the total uncoupling of this human enzyme. Moreover, measurements in the presence or absence of the substrate show that the substrate lowers the level of uncoupling only related to the H2O2 and not the superoxide radical. This is consistent with the entry point of the substrate in this enzyme's catalytic cycle. These findings highlight the importance of the involvement of hFMO3 in the production of radicals in the endoplasmic reticulum, as well as the relevance of single-nucleotide polymorphism leading to deleterious effects of oxidative stress.

Flavin-Containing Monooxygenase 3 Polymorphic Variants Significantly Affect Clearance of Tamoxifen and Clomiphene.

Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolising enzyme that oxygenates many drugs and xenobiotics in the liver. This enzyme is also known to exhibit single nucleotide polymorphisms (SNPs) that can alter the rates of monooxygenation of therapeutic agents. The purpose of this study was to investigate the effect of the three common polymorphic variants of hFMO3 (V257M, E158K and E308G) on the metabolism and clearance of three structurally similar compounds: tamoxifen (breast cancer medication), clomiphene (infertility medication) and GSK5182 (antidiabetic lead molecule). For GSK5182, none of the three variants showed any significant differences in its metabolism when compared to the wild-type enzyme. In the case of clomiphene, two of the variants, V257M and E308G, exhibited a significant increase in all the kinetic parameters measured with nearly two times faster clearance. Finally, for tamoxifen, a mixed behaviour was observed; E158K variant showed a significantly higher clearance compared to the wild type, whereas V257M mutation had the opposite effect. Overall, the data obtained demonstrate that there is no direct correlation between the SNPs and the metabolism of these three hFMO3 substrates. The metabolic capacity is both variant-dependent and substrate-dependent and therefore when testing new drugs or administering already approved therapies, these differences should be taken into consideration.