Methimazole assays were performed in triplicate on a single batch of microsomes for each heterologously expressed human FMO. four enzyme-catalyzed reactions are similar,kcatand, consequently,kcat/KM(the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa. Pulmonary tuberculosis (TB) is a serious respiratory disease caused by the opportunistic bacteriumMycobacterium tuberculosis. The World Health Organization estimated 9.2 million new cases of TB infection worldwide in 2006, of which 31% were in Africa. The appearance of strains ofM. tuberculosisthat are resistant to more than one first-line antitubercular drug has required the use of second-line drugs (Peloquin, 1993), such as the thiourea thiacetazone (TAZ; 4-formylacetanilide thiosemicarbazone) and the thioamide ethionamide (ETA; 2-ethylpyridine-4-carbothioamide). TAZ has been widely used in the developing world (Brown, 1992). Although an effective treatment for multidrug-resistant TB, it can produce adverse effects such as liver toxicity, gastrointestinal disturbances, and life-threatening skin Ciclopirox reactions, particularly in human immunodeficiency virus patients (Teklu, 1976;Brown, 1992;Peloquin, 1993;Ipuge et al., 1995), and, consequently, its use has been discontinued in several countries (Brown, 1992). ETA continues to be prescribed in both developed and developing countries. Both TAZ and ETA are prodrugs that are converted to their active forms by the mycobacterial Ciclopirox enzyme EtaA (Baulard et al., 2000;DeBarber et al., 2000;Qian and Ortiz de Montellano, 2006), a flavin-containing monooxygenase (FMO) (Vannelli et al., 2002). EtaA activates TAZ by two sequential oxidation steps to form a sulfinic acid and a carbodiimide via a postulated sulfenic acid intermediate (Qian and Ortiz de Montellano, 2006). TAZ treatment affects mycolic Ciclopirox acid biogenesis in mycobacteria (Alahari et al., 2007;Dover et al., 2007), and this may be the mechanism by which the drug exerts its antimicrobial effect. The FMOs (EC 1.14.13.8) of mammals catalyze the oxidative metabolism of numerous xenobiotics, including pesticides, fertilizers, and therapeutic drugs (Krueger and Williams, 2005;Cashman and Zhang, 2006;Phillips et al., 2007;Phillips and Shephard, 2008). Humans express five functional FMOs, FMOs 1 through 5 (Phillips et al., 1995;Hernandez et al., 2004). FMO1, FMO2, and FMO3 can bioactivate thiourea-based drugs (Smith and Crespi, 2002;Henderson et al., 2004;Onderwater et al., 2006), and FMO1 and FMO3 have been shown to catalyze oxygenation of TAZ in vitro, forming the same products as EtaA (Qian and Ortiz de Montellano, 2006). A genetic polymorphism of theFMO2gene, g.23238C>T (Q472X), gives rise to an allele,FMO2*2, which encodes a truncated, nonfunctional protein (FMO2.2) (Dolphin et al., 1998). Essentially all Europeans and Asians are homozygous forFMO2*2and thus Lep do not express functional FMO2 (Dolphin et al., 1998;Whetstine et al., 2000). However, Ciclopirox in sub-Saharan Africa, and in populations recently descended from this region, a substantial proportion of individuals possess at least one copy of the ancestralFMO2*1allele, which encodes a full-length functional protein (FMO2.1) (Dolphin et al., 1998;Whetstine et al., 2000;Veeramah et al., 2008). In contrast toFMO1andFMO3, which in the adult human are expressed primarily in kidney and liver, respectively (Dolphin et al., 1996;Yeung et al., 2000;Hernandez et al., 2004;Cashman and Zhang, 2006), the main site of expression ofFMO2is the lung (Dolphin et al., 1998;Krueger et al., 2002;Hernandez et al., 2004;Cashman and Zhang, 2006). Expression of functional FMO2.1 has been confirmed in lung microsomes from an individual heterozygous for theFMO2*1allele (Krueger et al., 2002). Because TAZ and ETA act against mycobacteria in the lung, we investigated the ability of human FMO2.1 to catalyze the oxygenation of these antitubercular drugs. In this article, we show that the protein encoded by theFMO2*1allele, FMO2.1, catalyzes oxygenation of both TAZ and ETA, forming the same metabolites as those produced by human FMO1 and FMO3 and by the mycobacterial enzyme EtaA. Furthermore, we show that the specificity constant of FMO2.1 for TAZ is higher than that of any of the other three enzymes. Our results provide a potential explanation.