Abstract
Human flavin-containing monooxygenase 3 (hFMO3) catalyses the oxygenation of a wide variety of compounds including drugs as well as dietary compounds. It is the major hepatic enzyme involved in the production of the N-oxide of trimethylamine (TMAO) and clinical studies have uncovered a striking correlation between plasma TMAO concentration and cardiovascular disease. Certain mutations within the hFMO3 gene cause defective trimethylamine (TMA) N-oxygenation leading to trimethylaminuria (TMAU) also known as fish-odour syndrome. In this paper, the inactivation mechanism of a TMAU-causing polymorphic variant, N61S, is investigated. Transient kinetic experiments show that this variant has a > 170-fold lower NADPH binding affinity than the wild type. Thermodynamic and spectroscopic experiments reveal that the poor NADP+ binding affinity accelerates the C4a-hydroperoxyFAD intermediate decay, responsible for an unfavourable oxygen transfer to the substrate. Steady-state kinetic experiments show significantly decreased N61S catalytic activity towards other substrates; methimazole, benzydamine and tamoxifen. The in vitro data are corroborated by in silico data where compared to the wild type enzyme, a hydrogen bond required for the stabilisation of the flavin intermediate is lacking. Taken together, the data presented reveal the molecular basis for the loss of function observed in N61S mutant.
Highlights
Human hepatic flavin-containing monooxygenase isoform 3 catalyses the oxygenation of a wide variety of nitrogen- and sulphur-containing chemicals including drugs as well as dietary compounds resulting in polar metabolites which are readily excreted[1,2]
Kinetic parameters of reductive and re-oxidative half-reactions were determined spectroscopically by stopped-flow and the binding properties of NADP+ to Human flavin-containing monooxygenase 3 (hFMO3) were characterized by isothermal titration calorimetry (ITC), circular dichroism (CD), differential scanning calorimetry (DSC) and trypsin digestion
The N61S variant of hFMO3 was expressed in E. coli and purified by Ni-affinity chromatography by protocols previously described for the wild type enzyme[20,21], resulting in a yield of 11 mg of the purified protein
Summary
Human hepatic flavin-containing monooxygenase isoform 3 (hFMO3) catalyses the oxygenation of a wide variety of nitrogen- and sulphur-containing chemicals including drugs as well as dietary compounds resulting in polar metabolites which are readily excreted[1,2]. In the reductive half-reaction, the FAD cofactor is first reduced through a hydride ion transfer from the nicotinamide C4 atom of NADPH to the flavin N5 atom which subsequently reacts with molecular oxygen to yield the C4a-hydroperooxyFAD intermediate[3,4,5]. In spite of growing knowledge of genetic polymorphisms of hFMO3 and those associated with trimethylaminuria, the molecular mechanisms underlying these loss-of-function genetic variants is still unclear due to lack of detailed structural information for hFMO3 as well as availability of pure and holo-protein. Poor NADP+ binding affinity of the mutant leads to an unstable C4a-hydroperoxyFAD intermediate, abolishing or decreasing the catalytic efficiency of hFMO3 towards its substrates
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