Vitamin B12 is a complex organometallic cofactor associated with three subfamilies of enzymes: the adenosylcobalamin-dependent isomerases, the methylcobalamin-dependent methyltransferases, and the dehalogenases. Different chemical aspects of the cofactor are exploited during catalysis by the isomerases and the methyltransferases. Thus, the cobalt-carbon bond ruptures homolytically in the isomerases, whereas it is cleaved heterolytically in the methyltransferases. The reaction mechanism of the dehalogenases, the most recently discovered class of B12 enzymes, is poorly understood. Over the past decade our understanding of the reaction mechanisms of B12 enzymes has been greatly enhanced by the availability of large amounts of enzyme that have afforded detailed structure-function studies, and these recent advances are the subject of this review.

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The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes.

Radical catalysis in coenzyme B12-dependent isomerization (eliminating) reactions.

SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.

Diverse Bacterial Microcompartment Organelles

Salmonellae can use ethanolamine (EA) as a sole source of carbon and nitrogen. This ability is encoded by an operon (eut) containing 17 genes, only 6 of which are required under standard conditions (37°C; pH 7.0). Five of the extra genes (eutM, -N, -L, -K, and -G) become necessary under conditions that favor loss of the volatile intermediate, acetaldehyde, which escapes as a gas during growth on EA and is lost at a higher rate from these mutants. The eutM, -N, -L, and -K genes encode homologues of shell proteins of the carboxysome, an organelle shown (in other organisms) to concentrate CO2. We propose that carboxysome-like organelles help bacteria conserve certain volatile metabolites—CO2 or acetaldehyde—perhaps by providing a low-pH compartment. The EutG enzyme converts acetaldehyde to ethanol, which may improve carbon retention by forming acetals; alternatively, EutG may recycle NADH within the carboxysome.

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Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica.

Glycerol and diol dehydratases are inducible, coenzyme B12-dependent enzymes found together in Klebsiella pneumoniae ATCC 25955 during anaerobic growth on glycerol. Mutants of this strain isolated by a novel procedure were separately constitutive for either dehydratase, showing the structural genes for the two enzymes to be under independent control in vivo. Glycerol dehydratase and a trimethylene glycol dehydrogenase were implicated as members of a pleiotropic control system that includes glycerol dehydrogenase and dihydroxyacetone kinase for the anaerobic dissimilation of glycerol (the "dha system"). The dehydratase and dehydrogenases were induced by dihydroxyacetone and were jointly constitutive in mutants isolated as constitutive for either the dha system or glycerol dehydratase. These data and the stimulation of growth by Co2+ suggested that glycerol dehydratase and trimethylene glycol dehydrogenase are obligatory enzymes for anaerobic growth on glycerol as the sole carbon source. Images

Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases.

Substrate specificity of coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator.

Coenzyme action of adenosyl-13-epicobalamin in the diol dehydrase system

Mechanism of inhibition of Chromatium D growth by L-methionine regulation of L-threonine biosynthesis by the intracellular level of S-adenosylmethionine

l-Methionine strongly inhibited the growth of Chromatium D when added at a low concentration to the growth medium containing both sulfide and thiosulfate. S-Adenosyl-l-methionine inhibited the growth, irrespective of the coexistence of sulfide and thiosulfate. Upon addition of l-methionine to the growth media, the presence of both sulfide and thiosulfate in the media stimulated the in vivo conversion of l-methionine to S-adenosyl-l-methionine, and consequently increased the intracellular level of S-adenosyl-l-methionine. From these data, it was strongly suggested that the unusual accumulation of S-adenosyl-l-methionine in the cells of Chromatium D is responsible for the growth inhibition by l-methionine.The level of S-adenosyl-l-methionine synthetase (ATP: l-methionine S-adenosyltransferase, EC 2.5.1.6) was significantly enhanced by adding l-methionine, sulfide and thiosulfate to the growth medium.

Tadahiro Shirakashi

Papers

Substrate specificity of coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator.

Coenzyme action of adenosyl-13-epicobalamin in the diol dehydrase system

Mechanism of inhibition of Chromatium D growth by L-methionine regulation of L-threonine biosynthesis by the intracellular level of S-adenosylmethionine

Growth inhibition of Chromatium D by L-methionine and its correlation to unusual accumulation of S-adenosyl-L-methionine in the cell.