catalysts Review Endogenous Roles of Mammalian Flavin-Containing Monooxygenases Ian R. Phillips 1,2,* and Elizabeth A. Shephard 1 1 Department of Structural and Molecular Biology, University College London, London WC1E 6BT, UK; [email protected] 2 School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, UK * Correspondence: [email protected] Received: 1 November 2019; Accepted: 25 November 2019; Published: 28 November 2019 Abstract: Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of numerous foreign chemicals. This review considers the roles of FMOs in the metabolism of endogenous substrates and in physiological processes, and focuses on FMOs of human and mouse. Tyramine, phenethylamine, trimethylamine, cysteamine, methionine, lipoic acid and lipoamide have been identified as endogenous or dietary-derived substrates of FMOs in vitro. However, with the exception of trimethylamine, the role of FMOs in the metabolism of these compounds in vivo is unclear. The use, as experimental models, of knockout-mouse lines deficient in various Fmo genes has revealed previously unsuspected roles for FMOs in endogenous metabolic processes. FMO1 has been identified as a novel regulator of energy balance that acts to promote metabolic efficiency, and also as being involved in the biosynthesis of taurine, by catalyzing the S-oxygenation of hypotaurine. FMO5 has been identified as a regulator of metabolic ageing and glucose homeostasis that apparently acts by sensing or responding to gut bacteria. Thus, FMOs do not function only as xenobiotic-metabolizing enzymes and there is a risk that exposure to drugs and environmental chemicals that are substrates or inducers of FMOs would perturb the endogenous functions of these enzymes. Keywords: cholesterol; energy balance; FMO; glucose; human; hypotaurine; insulin; mouse; taurine; weight 1. Introduction Flavin-containing monooxygenases (FMOs; EC 1.14.13.8) are best known for their role in catalyzing the oxidative metabolism of numerous foreign chemicals, which include therapeutic drugs and environmental pollutants [1–4]. This review, however, considers the roles of FMOs in the metabolism of endogenous substrates and in physiological processes. It focuses on FMOs of human and mouse and how the use of genetically modified mouse models are contributing to our understanding of the endogenous roles of FMOs. The human genome encodes five functional FMO genes, designated FMO1, 2, 3, 4 and 5 [5–7]. Four of these genes, FMOs 1, 2, 3 and 4 are located within a 245-kb cluster on chromosome 1, in the region q24.3 [7]. An additional FMO gene, FMO6P, located within this cluster, does not produce a correctly processed mRNA and, thus, is classified as a pseudogene [8]. The FMO5 gene is located closer to the centromere in the region 1q21.1 [7]. A second cluster of FMO genes, located at 1q24.2, consists of five pseudogenes, designated FMO7P, 8P, 9P, 10P and 11P [7]. In the mouse genome, five Fmo genes, Fmo1, 2, 3, 4 and 6, which are the orthologues of the corresponding human genes, are similarly clustered on Chromosome 1 [7]. The Fmo5 gene, which is orthologous to human FMO5, is located on mouse Chromosome 3 [7]. On mouse Chromosome 1 ~3.5 Mb from the main Fmo gene cluster there is a second cluster of Fmo genes containing three genes, Fmo9, Fmo12 and Fmo13 [7]. Based on Catalysts 2019, 9, 1001; doi:10.3390/catal9121001 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 1001 2 of 19 their sequences, Fmo6, Fmo9, Fmo12 and Fmo13 appear to be functional genes; however, the functional capabilities of their protein products have not been analysed. 2. Developmental Stage- and Tissue-Specific Regulation of Expression of FMOs in Human and Mouse In both human and mouse, each FMO gene has a distinct pattern of developmental stage- and tissue-specific expression. However, there are marked differences between the two species (Table1). Adult humans do not express FMO1 in liver because in this tissue expression of FMO1 is switched off after birth [9–11]. Transfection experiments revealed that LINEs (long-interspersed nuclear element)-1 like elements located upstream of the core hepatic promoter of the human FMO1 gene repress transcription of the gene [12]. Expression of FMO1 in human extra-hepatic tissues such as kidney [10,13,14] and small intestine [13] is under the control of alternative downstream promoters P1 and P2 [7,12]. In contrast, in mouse the Fmo1 gene is highly expressed in adult liver [15,16]. The gene is also expressed in several other tissues in mouse, including kidney, lung [16], white adipose tissue [17] and brain, a tissue in which it is the most highly expressed FMO [16]. Table 1. Major sites of expression of flavin-containing monooxygenases (FMOs) in adult human and mouse tissues. Human Mouse Male Mouse Female FMO1 Kidney Liver, Lung, Kidney Liver, Lung, Kidney FMO2 Lung Lung Lung FMO3 Liver - Liver FMO5 Liver, Gastrointestinal tract Liver, Gastrointestinal tract Liver, Gastrointestinal tract The main site of expression in both human and mouse of the gene encoding FMO2 is the lung [2,16,18] (Table1). Most humans, however, are homozygous for a genetic variant of FMO2, c.1414C > T[p.(Gln472*)], which introduces a premature translation stop codon. The resulting allele, FMO2*2A, encodes a polypeptide of 471 amino-acid residues that lacks the C-terminal 64 residues and is catalytically inactive [19]. Some individuals of recent African descent possess the ancestral allele, FMO2*1, which encodes a full-length polypeptide of 535 amino-acid residues (FMO2.1) that is catalytically active [19]. In sub-Saharan Africa the ancestral gene is widespread and can attain a frequency of up to 26% in some regions, with up to 50% of individuals possessing at least one FMO2*1 allele [20]. The liver is the main site of expression of FMO3 in humans [10] (Table1). It is also expressed in skin [21], pancreas and in adrenal medulla and cortex [7]. In the liver, FMO3 gene expression is activated within the first two years after birth and increases to reach a maximum in adulthood [11]. Apart from a decline during menstruation [22,23], the expression of FMO3 is similar in females and males. In mouse, however, there is a marked difference in expression of the Fmo3 gene in liver of females and males [16,24]: in females the gene is expressed throughout adulthood, whereas in males, the expression declines from three weeks of age and is undetectable by five to six weeks of age. In contrast, in lung the Fmo3 gene is expressed in both genders throughout adulthood [16]. The expression of FMO4 is very low in both human and mouse [2,7,10,16]. In both human and mouse the gene encoding FMO5 is most highly expressed in the liver [2,16] (Table1). The gene is also expressed in both species in the intestinal tract including the stomach and small and large intestine [2,7,25,26]. In humans, the gene encoding FMO5 is also expressed in pancreas [7] and in mouse in the kidney [16]. Catalysts 2019, 9, 1001 3 of 19 3. Catalytic Mechanism Our understanding of the catalytic mechanism of FMOs is based on the early work of Dan Catalysts 2019, 9, x FOR PEER REVIEW 3 of 19 Ziegler and colleagues [27–29]. FMOs contain FAD, as a prosthetic group, and, for catalysis, require NADPHoxygenation, as a cofactor a soft and nucleophilic oxygen as heteroatom, a co-substrate. typically Preferred nitrogen substrates or sulfur, contain, but as in their some site ofcases oxygenation,phosphorusa soft or selenium.nucleophilic The heteroatom, mechanism typically of action nitrogen of FMOs or is sulfur, unusual but and in some differs cases in several phosphorus aspects or selenium.from other The monooxygenases mechanism of actionsuch as of cytochromes FMOs is unusual P450 [30]. and diFMOsffers incan several activate aspects oxygen from in the other form monooxygenasesof a stable C4a such‐hydroperoxyflavin as cytochromes P450in the [30 absence]. FMOs of can bound activate substrate oxygen (Figure in the form 1). FMOs of a stable accept C4a-hydroperoxyflavinelectrons directly from in thetheir absence bound of co bound‐factor, substrate NADPH, (Figure and do1). not FMOs require accept an electronsaccessory directly protein to fromassist their in bound the oxygenation co-factor, NADPH, of a substrate. and do not FMOs require oxygenate an accessory substrates protein via to assista two in‐electron the oxygenation mechanism of athat substrate. usuallyFMOs results oxygenate in the productionsubstrates of viareadily a two-electron excretable detoxification mechanism that products. usually results in the production of readily excretable detoxification products. Figure 1. Representation of the catalytic cycle of mammalian FMOs. 1. NADPH binds to the enzyme and reduces FAD to FADH2. 2. Molecular oxygen binds and is reduced, forming C4a-hydroperoxyflavin, whichFigure is stabilized 1. Representation by NADP +of. 3.the Substrate catalytic (S)cycle is oxygenated of mammalian leaving FMOs. the 1. prosthetic NADPH group binds into thethe formenzyme of C4a-hydroflavin.and reduces FAD 4. Water to FADH is released,2. 2. andMolecular FAD is reformed.oxygen binds 5. NADP and+ isis released. reduced, Uncoupling forming ofC4a‐ thehydroperoxyflavin, cycle produces hydrogen which is peroxide stabilized from by NADP the C4a-hydroperoxyflavin,+. 3. Substrate (S) is oxygenated and NADP leaving+ is released the prosthetic to reformgroup FAD in the (dashed form lines).of C4a‐ Basedhydroflavin. on [29, 314. ].Water is released, and FAD is reformed. 5. NADP+ is released. Uncoupling of the cycle produces hydrogen peroxide from the C4a‐hydroperoxyflavin, and NADP+ Theis rate-limiting released to reform steps FAD of FMO-catalyzed (dashed lines). Based reactions on [29,31]. are the breakdown of FADH-OH to release water and the release of NADP+. As both of these steps occur after the oxygenation of substrate, the structureThe rate of‐ thelimiting substrate steps usuallyof FMO‐ hascatalyzed little ereactionsffect on theare catalyticthe breakdown constant of FADH (kcat) and‐OH the to releaseKM + forwater the substrate and the release is the key of NADP determinant.