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The methods of homologous high-level expression and simple large-scale purification for coenzyme B(12)-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer alpha(6)beta(6). Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B(12), the binding of 1 mol of the inhibitor per mol of the alphabeta unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme.

The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5'-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg(2+) and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5'-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H(2)S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.

Coenzyme B(12) serves as a cofactor for enzymatic radical reactions. The essential steps in all the coenzyme B(12)-dependent rearrangements are two hydrogen abstraction steps: hydrogen abstraction of the adenosyl radical from substrates, and hydrogen back-abstraction (recombination) of a product-derived radical from 5'-deoxyadenosine. The energetic feasibility of these hydrogen abstraction steps in the diol dehyratase reaction was examined by theoretical calculations with a protein-free, simplified model at the B3LYP/6-311G* level of density functional theory. Activation energies for the hydrogen abstraction and recombination with 1,2-propanediol as substrate are 9.0 and 15.1 kcal/mol, respectively, and essentially not affected by coordination of the substrate and the radical intermediate to K+. Since these energies can be considered to be supplied by the substrate-binding energy, the computational results with this simplified model indicate that the hydrogen abstraction and recombination in the coenzyme B(12)-dependent diol dehydratase reaction are energetically feasible.

Anaerobic respiration with elemental sulfur/polysulfide or organic disulfides is performed by several bacteria and archaea, but has only been investigated in a few organisms in detail. The electron transport chain that catalyzes polysulfide reduction in the Gram-negative bacterium Wolinella succinogenes consists of a dehydrogenase (formate dehydrogenase or hydrogenase) and polysulfide reductase. The enzymes are integrated in the cytoplasmic membrane with the catalytic subunits exposed to the periplasm. The mechanism of electron transfer from formate dehydrogenase or hydrogenase to polysulfide reductase is discussed. The catalytic subunit of polysulfide reductase belongs to the family of molybdopterin-dinucleotide-containing oxidoreductases. From the hyperthermophilic archaeon Pyrodictium abyssi isolate TAG11 an integral membrane complex has been isolated which catalyzes the reduction of sulfur with H2 as electron donor. This enzyme complex, which is composed of a hydrogenase and a sulfur reductase, contains heme groups and several iron-sulfur clusters, but does not contain molybdenum or tungsten. In methanogenic archaea, the heterodisulfide of coenzyme M and coenzyme B is the terminal electron acceptor of the respiratory chain. In methanogens belonging to the order Methanosarcinales, this respiratory chain is composed of a dehydrogenase, the membrane-soluble electron carrier methanophenazine, and heterodisulfide reductase. The catalytic subunit of heterodisulfide reductase contains only iron-sulfur clusters. An iron-sulfur cluster may directly be involved in the reduction of the disulfide substrate.

Radicals are reactive intermediates of growing importance in enzymatic catalysis. There are reactions in which neutral radicals participate and those where radical anions are involved. The former class is illustrated by lysine 2,3-aminomutase and also by enzymes dependent on coenzyme B12, that catalyse carbon skeleton rearrangements (e.g. glutamate mutase). A substrate-based radical for both lysine 2,3-aminomutase and glutamate mutase has been characterised by EPR spectroscopy. Representatives of the second class are 2-hydroxyglutaryl-CoA dehydratase, benzoyl-CoA reductase, DNA photolyase and chorismate synthase, all of which may generate the radical anion by one-electron reduction. 4-Hydroxybutyryl-CoA dehydratase, pyruvate formate lyase, and the coenzyme B12-dependent eliminases (ribonucleotide reductase, ethanolamine ammonia lyase and diol dehydratase) could be examples of radical anion formation by one-electron oxidation. The electron-rich ketyl-like radical anions cause the elimination of an adjacent group. The advantages of using radicals as intermediates in enzymatic transformations are their high reactivity and special properties. However, this reactivity includes rapid bimolecular combination with dioxygen and radicals are therefore primarily utilised as intermediates by anaerobic organisms.

The genes encoding coenzyme B 12 -dependent glycerol dehydratase of Clostridium pasteurianum were subcloned and expressed in Escherichia coli . The native molecular mass of the enzyme is 190 000 Da. The enzyme converts glycerol, 1,2-propanediol and 1,2-ethanediol to 3-hydroxypropionaldehyde, propionaldehyde and acetaldehyde, respectively, but glycerol is the preferred substrate. The nucleotide sequences of the dhaBCE genes encoding the three subunits of glycerol dehydratase and of orfZ whose function is unknown were determined. The deduced products of the dhaBCE genes with calculated molecular masses of 60 813, 19 549 and 16 722 Da, respectively, revealed high similarity to amino acid sequences of subunits of coenzyme B 12 -dependent glycerol and diol dehydratases from other organisms.

In a non-targeted analysis of thiol-containing compounds in the hyperthermophilic methanogen Methanocaldococcus jannashii , we discovered three unexpected metabolites: 3-mercaptopropionic acid (MPA), 2-hydroxy-4-mercaptobutyric acid (HMBA), and 4-mercapto-2-oxobutyric acid (MOB). HMBA and MOB have never been reported as natural products, while MPA is highly prevalent in aquatic environments as a result of biotic and abiotic processing of sulfur-containing compounds. This report provides evidence that HMBA and MOB are part of a biosynthetic pathway to generate MPA in M. jannaschii . We show that HMBA can be biosynthesized from malate semialdehyde and hydrogen sulfide, likely using a mechanism similar to that proposed for coenzyme M, coenzyme B, and homocysteine biosynthesis in methanogens, where an aldehyde is converted to a thiol. The L-sulfolactate dehydrogenase, derived from the MJ1425 gene, is shown to catalyze the NAD-dependent oxidation of HMBA to MOB. Finally, we demonstrate that HMBA can be used as a biosynthetic precursor to MPA in M. jannaschii cell extracts. This proposed pathway may contribute to the wide occurrence of MPA in marine environments and indicates that MPA must serve some important function in M. jannaschii .

Adenosylcobalamin (coenzyme B12) dependent glutamate mutase catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-methylaspartate. This is the first step of the fermentation of glutamate by the strict anaerobic bacterium Clostridium cochlearium. The enzyme consists of the two protein components E and S. The gene encoding component S (glmS) was cloned in Escherichia coli and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence showed very strong identities to the sequence of the glmS (also called mutS) gene (80%) and to component S (82%) from the related C. tetanomorphum, respectively. Cell-free extracts of E. coli carrying the glmS gene showed glutamate mutase activity which was strictly dependent on the addition of coenzyme B12 and component E purified from C. cochlearium. Enzyme activity of the recombinant protein was achieved up to 2200 nkat/g wet cells which is due to a ten-fold overexpression compared with the activities determined in cell-free extracts of C. cochlearium. This is the first report of overexpression of an active component of glutamate mutase. A rapid purification procedure consisting only of ammonium sulfate precipitation and a gel filtration step was developed to obtain large amounts of pure component S in a short time.