flavin-containing monooxygenase flavin-containing monooxygenase the flavin-containing monooxygenase (fmo) protein family specializes in the oxidation of xeno-substrates in order to facilitate the excretion of these compounds from living organisms. these enzymes can oxidize a wide array of heteroatoms, particularly soft nucleophiles, such as amines, sulfides, and phosphites. this reaction requires an oxygen, an nadph cofactor, and an fad prosthetic group. fmos share several structural features, such as a nadph binding domain, fad binding domain, and a conserved arginine residue present in the active site. recently, fmo enzymes have received a great deal of attention from the pharmaceutical industry both as a drug target for various diseases and as a means to metabolize pro-drug compounds into active pharmaceuticals. these monooxygenases are often misclassified because they share activity profiles similar to those of cytochrome p450 (cyp450), which is the major contributor to oxidative xenobiotic metabolism. however, a key difference between the two enzymes lies in how they proceed to oxidize their respective substrates; cyp enzymes make use of an oxygenated heme prosthetic group, while the fmo family utilizes fad to oxidize its substrates. flavin-containing monooxygenaseribbon diagram of yeast fmo (pdb: 1vqw).identifiersec no.1.14.13.8cas no.37256-73-8 databasesintenzintenz viewbrendabrenda entryexpasynicezyme viewkeggkegg entrymetacycmetabolic pathwaypriamprofilepdb structuresrcsb pdb pdbe pdbsumgene ontologyamigo / quickgosearchpmcarticlespubmedarticlesncbiproteins flavin-containing monooxygenase fmoidentifierssymbolflavin_moasepfampf00743interproipr000960membranome262available protein structures:pfam structures / ecod pdbrcsb pdb; pdbe; pdbjpdbsumstructure summary history prior to the 1960s, the oxidation of xenotoxic materials was thought to be completely accomplished by cyp450. however, in the early 1970s, dr. daniel ziegler from the university of texas at austin discovered a hepatic flavoprotein isolated from pig liver that was found to oxidize a vast array of various amines to their corresponding nitro state. this flavoprotein named "ziegler's enzyme" exhibited unusual chemical and spectrometric properties. upon further spectroscopic characterization and investigation of the substrate pool of this enzyme, dr. ziegler discovered that this enzyme solely bound fad molecule that could form a c4a-hydroxyperoxyflavin intermediate, and that this enzyme could oxidize a wide variety of substrates with no common structural features, including phosphines, sulfides, selenium compounds, amongst others. once this was noticed, dr. ziegler's enzyme was reclassified as a broadband flavin monooxygenase. in 1984, the first evidence for multiple forms of fmos was elucidated by two different laboratories when two distinct fmos were isolated from rabbit lungs. since then, over 150 different fmo enzymes have been successfully isolated from a wide variety of organisms. up until 2002, only 5 fmo enzymes were successfully isolated from mammals. however, a group of researchers found a sixth fmo gene located on human chromosome 1. in addition to the sixth fmo discovered as of 2002, the laboratories of dr. ian philips and elizabeth sheppard discovered a second gene cluster in humans that consists of 5 additional pseudogenes for fmo on human chromosome 1. evolution of fmo gene family the fmo family of genes is conserved across all phyla that have been studied so far, therefore some form of the fmo gene family can be found in all studied eukaryotes. fmo genes are characterized by specific structural and functional constraints, which led to the evolution of different types of fmo's in order to perform a variety of functions. divergence between the functional types of fmo's (fmo 1–5) occurred before the amphibians and mammals diverged into separate classes. fmo5 found in vertebrates appears to be evolutionarily older than other types of fmo's, making fmo5 the first functionally distinct member of the fmo family. phylogenetic studies suggest that fmo1 and fmo3 are the most recent fmo's to evolve into enzymes with distinct functions. although fmo5 was the first distinct fmo, it is not clear what function it serves since it does not oxygenate the typical fmo substrates involved in first-pass metabolism. analyses of fmo genes across several species have shown extensive silent dna mutations, which indicate that the current fmo gene family exists because of selective pressure at the protein level rather than the nucleotide level. fmo's found in invertebrates are found to have originated polyphyletically; meaning that a phenotypically similar gene evolved in invertebrates which was not inherited from a common ancestor. classification and characterization fmos are one subfamily of class b external flavoprotein monooxygenases (ec 1.14.13), which belong to the family of monooxygenase oxidoreductases, along with the other subfamilies baeyer-villiger monooxygenases and microbial n-hydroxylating monooxygenases. fmo's are found in fungi, yeast, plants, mammals, and bacteria. mammals developmental and tissue specific expression has been studied in several mammalian species, including humans, mice, rats, and rabbits. however, because fmo expression is unique to each animal species, it is difficult to make conclusions about human fmo regulation and activity based on other mammalian studies. it is likely that species-specific expression of fmo's contributes to differences in susceptibility to toxins and xenobiotics as well as the efficiency with excreting among different mammals. six functional forms of human fmo genes have been reported. however, fmo6 is considered to be a pseudogene. fmos 1–5 share between 50–58% amino acid identity across the different species. recently, five more human fmo genes were discovered, although they fall in the category of pseudogenes. fmo1, fmo2, fmo3, fmo4, fmo5, fmo6 yeast unlike mammals, yeast (saccharomyces cerevisiae) do not have several isoforms of fmo, but instead only have one called yfmo. this enzyme does not accept xenobiotic compounds. instead, yfmo helps to fold proteins that contain disulfide bonds by catalyzing o2 and nadph-dependent oxidations of biological thiols, just like mammalian fmo's. an example is the oxidation of glutathione to glutathione disulfide, both of which form a redox buffering system in the cell between the endoplasmic reticulum and the cytoplasm. yfmo is localized in the cytoplasm in order to maintain the optimum redox buffer ratio necessary for proteins containing disulfide bonds to fold properly. this non-xenobiotic role of yfmo may represent the original role of the fmo's before the rise of the modern fmo family of enzymes found in mammals. plants plant fmo's play a role in defending against pathogens and catalyze specific steps in the biosynthesis of auxin, a plant hormone. plant fmo's also play a role in the metabolism of glucosinolates. these non-xenobiotic roles of plant fmo's suggest that other fmo functions could be identified in non-plant organisms. structure crystal structures have been determined for yeast (schizosaccharomyces pombe) fmo (pdb: 1vqw) and bacterial (methylophaga aminisulfidivorans) fmo (pdb: 2xvh). the crystal structures are similar to each other and they share 27% sequence identity. these enzymes share 22% and 31% sequence identity with human fmos, respectively. channel and active site of bacterial fmo with bound nadph and fad (pdb: 2xvh). fmos have a tightly bound fad prosthetic group and a binding nadph cofactor. both dinucleotide binding motifs form rossmann folds. the yeast fmo and bacterial fmo are dimers, with each monomer consisting of two structural domains: the smaller nadph binding domain and the larger fad-binding domain. the two domains are connected by a double linker. a channel between the two domains leads to the active site where nadph binds both domains and occupies a cleft that blocks access to the flavin group of fad, which is bound to the large domain along the channel together with a water molecule. the nicotinamide group of nadph interacts with the flavin group of fad, and the nadph binding site overlaps with the substrate binding site on the flavin group. fmos contain several sequence motifs that are conserved across all domains: fad-binding motif (gxgxxg) fmo identifying motif (fxgxxxhxxxf/y) nadph-binding motif (gxsxxa) f/latgy motif arginine residue in the active site the fmo identifying motif interacts with the flavin of fad. the f/latgy motif is a sequence motif common in n-hydroxylating enzymes. the arginine residue interacts with the phosphate group of nadph. function reactions catalyzed by fmos. the general function of these enzymes is to metabolise xenobiotics. hence, they are considered to be xenobiotic detoxication catalysts. these proteins catalyze the oxygenation of multiple heteroatom-containing compounds that are present in our diet, such as amine-, sulfide-, phosphorus-, and other nucleophilic heteroatom-containing compounds. fmos have been implicated in the metabolism of a number of pharmaceuticals, pesticides and toxicants, by converting the lipophilic xenobiotics into polar, oxygenated, and readily excreted metabolites. substrate diversity