Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions. butyryl-CoA dehydrogenase ͉ electron transfer flavoproteins ͉ genome sequence ͉ Rnf-dependent energy conservation

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Some methanogenic and acetogenic microorganisms have the catalytic capability to cleave heterolytically the COO bond of methanol. To obtain insight into the elusive enzymatic mechanism of this challenging chemical reaction we have investigated the methanol-activating MtaBC complex from Methanosarcina barkeri composed of the zinc-containing MtaB and the 5-hydroxybenzimidazolylcobamide-carrying MtaC subunits. Here we report the 2.5-Å crystal structure of this complex organized as a (MtaBC) 2 heterotetramer. MtaB folds as a TIM barrel and contains a novel zinc-binding motif. Zinc(II) lies at the bottom of a funnel formed at the Cterminal ␤-barrel end and ligates to two cysteinyl sulfurs (Cys-220 and Cys-269) and one carboxylate oxygen (Glu-164). MtaC is structurally related to the cobalamin-binding domain of methionine synthase. Its corrinoid cofactor at the top of the Rossmann domain reaches deeply into the funnel of MtaB, defining a region between zinc(II) and the corrinoid cobalt that must be the binding site for methanol. The active site geometry supports a S N 2 reaction mechanism, in which the COO bond in methanol is activated by the strong electrophile zinc(II) and cleaved because of an attack of the supernucleophile cob(I)amide. The environment of zinc(II) is characterized by an acidic cluster that increases the charge density on the zinc(II), polarizes methanol, and disfavors deprotonation of the methanol hydroxyl group. Implications of the MtaBC structure for the second step of the reaction, in which the methyl group is transferred to coenzyme M, are discussed.conformational change ͉ methanol metabolism ͉ x-ray structure ͉ zinc M ethanol is an abundant C 1 -compound in nature. Its major source is probably the plant cell wall component pectin from which methanol is released upon hydrolytic degradation. In oxic environments methanol is rapidly metabolized to CO 2 by aerobic methylotrophic microorganisms, which thereby prevents autooxidation to the cytotoxic formaldehyde (1). Methanol also does not accumulate in anaerobic environments, where methanogenic archaea (2) and acetogenic bacteria (3) reduce the C 1 -compound to methane, carbonylate it to acetate, and/or oxidize it to CO 2 in their energy metabolism.Methanol metabolism is initiated in methanogenic archaea by its reaction with coenzyme M (HS-CoM) to form methyl-coenzyme M (CH 3 -S-CoM) (reaction 1) (4, 5). Methyl-coenzyme M is the central intermediate for reduction to methane and oxidation to CO 2 .

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