Flavin-containing monooxygenase 3 (FMO3) is known primarily as an enzyme involved in the metabolism of therapeutic drugs. On a daily basis, however, we are exposed to one of the most abundant substrates of the enzyme trimethylamine (TMA), which is released from various dietary components by the action of gut bacteria.FMO3 converts the odorous TMA to nonodorous TMA N-oxide (TMAO), which is excreted in urine. Impaired FMO3 activity gives rise to the inherited disorder primary trimethylaminuria (TMAU). Affected individuals cannot produce TMAO and, consequently, excrete large amounts of TMA. A dysbiosis in gut bacteria can give rise to secondary TMAU. Recently, there has been much interest in FMO3 and its catalytic product,TMAO, because TMAO has been implicated in various conditions affecting health, including cardiovascular
disease, reverse cholesterol transport, and glucose and lipid homeostasis. In this review, we consider the dietary components that can give rise to TMA,the gut bacteria involved in the production of TMA from dietary precursors, the metabolic reactions by which bacteria produce and use TMA, and the enzymes that catalyze the reactions. Also included is information on bacteria that produce TMA in the oral cavity and vagina, two key microbiome niches that can influence health. Finally, the importance of the TMA/TMAO microbiome-host axis in health and disease, considering factors that affect bacterial production and host metabolism of TMA, the involvement of TMAO and FMO3 in disease, and the implications of the host-microbiome axis for management of TMAU.
Flavin-containing monooxygenases (FMOs) catalyse the NADPH-dependent oxidative metabolism of a wide array of foreign chemicals, including drugs, dietary-derived compounds, and environmental pollutants Humans possess five functional FMO genes: FMO1, 2, 3, 4, and 5 The main site of expression of FMO3 is the liver; however, high expression levels have also been observed in the skin of certain individuals The FMO3 gene is switched on in human liver at birth and can take several years for the gene to attain maximum expression as the liver develops to its full functional capacity FMO3 has an important relationship with the gutmicrobiome,with an abundant non drug substrate, trimethylamine(TMA), being derived from dietary components by the action of gut microbes.TMA is rapidly absorbed and is converted in the liver to trimethylamine N-oxide (TMAO). Of the five functional FMOs of humans (FMOs 1–5), only FMO3 effectively catalyzes the conversion of TMA to TMAO.FMO3 is thus an excellent example of a protein that participates in host-gut microbiome metabolic interaction. Rare genetic variants of the FMO3 gene that abolish or severely impair activity of the enzyme give rise to the inherited disorder primary trimethylaminuria (TMAU) (Phillips and Shephard, 2008) because of inefficient conversion of microbiome-derived odorous TMA to no odorous TMAO. Recently, there has been much interest in FMO3 and its catalytic product TMAO. This is because TMAO has been implicated in various TMA is an indicator of food spoilage, particularly of fish and milk. As early as the 1930s Beatty showed that TMA was produced during fish spoilage. The TMA is derived from bacterially mediated reduction of TMAO, which is present in large quantities in marine fish. The requirement of bacterial action for the production of TMA from the diet in vivo is demonstrated by the failure of germ-free rats fed carnitine , or of germ-free or antibiotic-treated mice fed a normal diet, to excrete TMA in their urine. TMA is derived from the diet by microbial degradation of precursors found in,for example, marine fish,eggs,offal,soyabeans,peas,and red meat. Once liberated, TMA is rapidly absorbed through the gut wall and transported to the liver, where it is converted to no odorous TMAO, which is then rapidly cleared in the urine. Analysis in vitro of heterologous expressed FMOs revealed that, at physiologic pH, N-oxygenation of TMA is catalysed by FMO3, with a KM of 28 mM and an apparent kcat of.30minutes21.Other FMOs are far less effective in catalyzingthis reaction: FMO1, FMO2, and FMO4,none of which is expressed in adult human liver, exhibit apparent kcats of0.1,1.0,and0.1minute21,respectively,where as FMO5,theonly other form of FMO expressed in the adult human liver (Phillips et al., 2007), is unable to catalyse the reaction. FMO1, which is expressed in human kidney,cancatalyze TMA N-oxygenation, with an apparent kcat of 5 minutes21, but only at substrate concentrations of 5mM),which are unlikely to be relevant for metabolism of TMA invivo.The importance of FMO3 for the metabolism of TMA in vivo is demonstrated by the marked reduction in the ability to N-oxygenate TMA of individuals homozygous or compound heterozygous for mutations that severely impair FMO3 activity. In addition to N-oxygenation, rat liver microsomes carry out demethylation of TMA, but the latter reaction is much less efficient. Demethylation of TMA in rat liver was show to be catalysed by a different FMO from that which catalyses production of the N-oxide. This FMO is probably FMO1, whose gene is expressed in the liver of rodents and other mammals, but not in adult human liver .In human volunteers, the demethylation product was found in low amounts and only in those dosed with high amounts of TMA (Al-Waiz, et al., 1987b). Consequently, in humans FMO3-catalyzed N-oxygenation is by far the most important route of metabolism of TMA. Since microbial diversity studies have entered the era of high through put sequencing and in silico analysis, more insight has been gained into the composition of the human gut microbiota; however, studies based on bacterial cultivation are still the main source for information about the metabolic capacities of the microbiota. Combining what is being uncovered about the composition of the human microbiota with what is known of the functional activity of its components enables identification of microbial metabolic pathways by which TMA can be produced or metabolized in the human gut.
TMAU, a Deficiency of FMO3 In humans, mutations that abolish or severely impair the activity of FMO3 cause the inherited disorder primary TMAU. Affected individuals have a severely reduced ability to convert TMA to TMAO and, consequently, excrete large amounts of odorous TMA in their urine, sweat, and breath. Although the disorder has no overt physiologic effects on patient health, it can have profound psychologic and social consequences, resulting in as severe loss of quality of life, in extreme cases giving rise to clinical depression and suicidal tendencies. Primary trimethylaminuria. In terms of drug metabolism, TMAU individuals have impaired metabolism of the FMO3 drug substrate benzydamine. Since the first discovery of a mutation known to cause TMAU A catalog of variants of the FMO3 gene and their effect on the ability of FMO3 to catalyse the oxygenation of TMA and drug substrates of the enzyme can be accessed at the FMO3 locus-specific mutation database. Production of large amounts of TMA, as a result of gut microbialaction, exacerbates the symptoms of primary TMAU. Inaddition, overproduction of TMA, as a consequence of adysbiosis of the gutmicrobiome, can give rise to a nongenetic form of the disorder, known as secondary TMAU. Therefore, a better understanding of the bacterial species that produce TMA in the gut may provide insights into why some individuals develop secondary TMAU in the absence of impaired N-oxygenation of TMA and offers the potential to develop improved strategies for the management and treatment of both primary and secondary forms of the disorder.
Consumed TMAO is not metabolized and passes through the body unchanged to be excreted in the urine. In the human gut, there main of the TMAO is reduced to TMA by bacterial TMAO reductase. Choline is an essential dietary nutrient, which can function as a precursor for the synthesis of phospholipids, including phosphatidylcholine, that are essential components of membranes, and of the neurotransmitteracetylcholine.Itispresentinhighquantitiesinavariety of foodstuffs, for example, beef liver, cauliflower, and peanuts , and high concentrations of free choline are present in human milk and in soya bean–derived milk formulae ). Choline is usually ingested as lecithin (also known as phosphatidylcholine), however, in which the choline moiety is covalently bound to a phosphatide (a phosphoglycerol attached to two fatty acids). Interconversion of lecithin and choline is bidirectional, conversion of lecithin to choline being catalysed by phospholipase D and the first step in the conversion of choline to lecithin by choline kinase. Free choline is absorbed throughout the small intestine and is subsequently integrated into cell membranes or actively taken up by the liver, where it can be converted to betaine, phospho choline, or lecithin. High amounts of choline may exceed the absorptive capacity and pass through to the large intestine, however, where it is metabolized to methylamines by microbial action).Choline is aquaternary ammonium compound containing a trimethylammonium moiety. Thus, it can act as a precursor for TMA . The bacterial conversion of choline to TMA involves the cleavage of the carbon-nitrogen bond of choline, producing TMA and acetaldehyde proposed that a glycyl radical enzyme Cut C,encoded by the bacterial choline utilization gene cluster (cut), might act as a choline TMA-lyase to catalyse this initial step in choline degradation. This was confirmed by demonstrating that deletion of cut C in Desulfovibrio desulfuricans abolished the ability of the organism to produce TMA from choline. Bioinformatics analysis revealed cutC homologs in 89 bacterial genomes. The homologs are not distributed evenly among the major bacterial phyla of the human gut, being present in Firmicutes, Actinobacteria, andProteobacteria spp. but absent from Bacteroidetes.Bacteria known to be associated with formation of TMA via choline degradation. Carnitine. Carnitine plays a key role in metabolism, being involved in thetransportoflong-chainfattyacids from the cytosolintomitochondria. Carnitine is a quaternary ammonium compound that can be synthesized in the body from methionine and lysine, with its immediate precursor being g-butyrobetaine. L-carnitine is present in red meat and dairy products, and it is estimated that the average nonvegetarian American consumes 100– 300mg/day.Of dietary carnitine, about half is absorbed from the intestine,and the other half is metabolized by gut flora, eventually resulting in the excretion of TMAO and g-butyrobetaine in urine and feces, respectively .Gut bacteria are thought to cleave the 3-hydroperoxybutyryl moiety from L-carnitine to produce TMA. This pathway has been observed in several bacteria, including Serratia marascescens and Acinetobacter calcoaceticus. Using bioinformatics approaches, identified a two component oxygenase/reductase Rieske-type enzyme, encoded by cntAB, that catalyzed the formation of TMA from carnitin