Mechanism of action of a flavin-containing monooxygenase

Abstract

Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein–cofactor and protein–substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein–cofactor and protein–substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4α-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.

Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population

“…A molecular modeling of human FMO3 (532 amino acids) is worth examining to understand the contribution of C-terminus based on the recently reported crystal structure of yeast FMO (447 amino acid) [31], in spite of the low sequence identity (~20%) and short length (~80%) of the whole human FMO3 structure.…”

Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria

“…Twenty picomoles of holo-FMO3 were used in initial studies with incubation times ranging from [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] minutes. Metabolic reactions with enzymes that exhibited low substrate turnover (N61S, M66I, P153L, and R492W) were repeated using 50-100 pmol of FMO for 20 minutes to facilitate product quantitation.…”

“…When the structure of FMO from Schizosaccharomyces pombe became available (PDB ID codes 2GVC (with FAD and substrate bound) and 2GV8 (with FAD and NADP bound) [20], the sequences of human FMO1, FMO2, and FMO3 (SwissProt Data Bank accessed via the ExPASY web site (http://www.expasy.org) [26] were added to the previously published alignment of S. pombe FMO, Saccharomyces cerevisiae FMO1 and Drosophila FMO2 (see Fig 8 of [20]). This alignment was used to guide construction of a homology model for FMO3 using the Molecular Operating Environment software, (MOE, available from the Chemical Computing Group, Montreal (http://www.chemcomp.com), by iteratively building and readjusting the alignment according to where insertions and deletions were deemed most likely to occur relative to yeast FMO.…”

“…A one residue Gly insertion in a loop (368-379) apposing the loop containing N61 causes the local geometry of the model to differ from the yeast structure. It is possible that this could have some bearing on enzyme reactivity, as this loop contains the reportedly [20] characteristic FMO identifying sequence, FxGxxxHxxxY/F, which in human FMO3 appears as VxGxxxSxGxxI.…”

“…This knowledge gap has been exacerbated by the lack of any detailed structural information for the microsomal FMOs. However, recently the crystal structure of a yeast FMO was solved [20] providing, for the first time, a highly useful template for construction of homology models for human FMO3. Therefore, the goal of the present study was to express disease-related genetic variants of FMO3, assess proper folding of the enzymes through analysis of FAD incorporation/ retention, and measure steady-state kinetic parameters for oxidation of the probe substrates trimethylamine, benzydamine, methyl p-tolyl sulfide (Figure 1).…”