Inactivation mechanism of N61S mutant of human FMO3 towards trimethylamine

• Published in: Scientific reports 2017-11, Vol.7 (1), p.14668-11, Article 14668
• Main Authors: Gao, Chongliang, Catucci, Gianluca, Castrignanò, Silvia, Gilardi, Gianfranco, Sadeghi, Sheila J.
• Format: Article
• Language: English
• Subjects: 631/1647; 631/45; 82; 82/83; Affinity; Computer Simulation; Dimethylaniline monooxygenase (N-oxide-forming); Enzymes; Experiments; Flavin; Humanities and Social Sciences; Humans; In Vitro Techniques; Metabolism, Inborn Errors - genetics; Metabolism, Inborn Errors - metabolism; Methylamines - metabolism; Methylamines - urine; multidisciplinary; Mutation; NADP; Odor; Oxidation-Reduction; Oxygenases - genetics; Oxygenases - metabolism; Oxygenation; Science; Science (multidisciplinary); Tamoxifen; Trimethylamine
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-15224-9

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.