The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL͞ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one (1), which supply nicotinate as a nitrogen, carbon, and energy source to a diverse set of dedicated nicotinatecatabolizing microorganisms (2). Nicotinate catabolism in all organisms starts with hydroxylation to 6-hydroxynicotinate by the well characterized and industrially used enzyme nicotinate dehydrogenase (3). Further catabolism depends on the availability of oxygen in the environment. In several aerobic organisms, such as Pseudomonads, 6-hydroxynicotinate is oxidatively decarboxylated to 2,5-dihydroxypyridine (4) or, in the unique case of Bacillus niacini, subjected to a second hydroxylation yielding 2,6-dihydroxynicotinate (5). Under microaerobic (6) or fermentative conditions (7), ferredoxin-dependent reduction to 1,4,5,6-tetrahydro-6-oxonicotinate (THON) is observed.Work by Harary (8) and Stadtman (9) identified an anaerobic soil bacterium now called Eubacterium barkeri (order Clostridiales) that fermented nicotinate according to the following equation:Cell extracts incubated with radioactively labeled nicotinate allowed a number of unusual intermediates to be identified (10, 11), and it became clear that the pathway was remarkably complex (see Fig. 1). Based on the identified intermediates, several anticipated enzymes were purified and characterized: nicotinate dehydrogenase (12), 6-hydroxynicotinate reductase (7), 2-methyleneglutarate mutase, and 3-methylitaconate isomerase (13,14). These findings outlined the nicotinate fermentation pathway and placed the identified intermediates in an enzymatic framework. The nicotinate dehydrogenase contains [2Fe-2S] clusters (15), FAD and molybdopterin cytosine dinucleotide (16), and has an unusual subunit composition [50,37,33, and 23 kDa (17)]. It has labile (nonselenocysteine) selenium (18) also identified in purine dehydrogenase from Clostridium purinolyticum and xanthine dehydrogenases from C. purinolyticum (19), Clostridium acidiurici (20), and E. barkeri (21). The selenium coordinates molybdenum (15) and is thought to be a selenido equivalent of the cyanolyzable sulfido-ligand (22) in the xanthine d...