adeninylpentylcobalamin and cobamamide

N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase beta-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83-93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. The enzyme exists as a trimer of the (alphabeta)(2) dimer. The active site is in the (beta/alpha)(8) barrel of the alpha-subunit; the beta-subunit covers the lower part of the cobalamin that is bound in the interface of the alpha- and beta-subunits. The structure complexed with adeninylpentylcobalamin revealed the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Argalpha(160) contributes to substrate binding most likely by hydrogen bonding with the O1 atom. The modeling study implies that marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme Co-C bond. The coenzyme adenosyl radical in the productive conformation was modeled by superimposing its adenine ring on the adenine ring-binding site followed by ribosyl rotation around the N-glycosidic bond. A major structural change upon substrate binding was not observed with this particular enzyme. Glualpha(287), one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co-C bond.

Topics: Catalytic Domain; Cobamides; Escherichia coli; Escherichia coli Proteins; Ethanolamine Ammonia-Lyase; Hydrogen Bonding; Organometallic Compounds; Protein Binding; Protein Subunits; Substrate Specificity; Vitamin B 12

Adenosylcobalamin (coenzyme B(12)) serves as a cofactor for enzymatic radical reactions. The adenosyl radical, a catalytic radical in these reactions, is formed by homolysis of the cobalt-carbon bond of the coenzyme, although the mechanism of cleavage of its organometallic bond remains unsolved.. We determined the three-dimensional structures of diol dehydratase complexed with adeninylpentylcobalamin and with cyanocobalamin at 1.7 A and 1.9 A resolution, respectively, at cryogenic temperatures. In the adeninylpentylcobalamin complex, the adenine ring is bound parallel to the corrin ring as in the free form and methylmalonyl-CoA-mutase-bound coenzyme, but with the other side facing pyrrole ring C. All of its nitrogen atoms except for N(9) are hydrogen-bonded to mainchain amide oxygen and amide nitrogen atoms, a sidechain hydroxyl group, and a water molecule. As compared with the cyanocobalamin complex, the sidechain of Seralpha224 rotates by 120 degrees to hydrogen bond with N(3) of the adenine ring.. The structure of the adenine-ring-binding site provides a molecular basis for the strict specificity of diol dehydratase for the coenzyme adenosyl group. The superimposition of the structure of the free coenzyme on that of enzyme-bound adeninylpentylcobalamin demonstrated that the tight enzyme-coenzyme interactions at both the cobalamin moiety and adenine ring of the adenosyl group would inevitably lead to cleavage of the cobalt-carbon bond. Rotation of the ribose moiety around the glycosidic linkage makes the 5'-carbon radical accessible to the hydrogen atom of the substrate to be abstracted.

Topics: Bacterial Proteins; Binding Sites; Cobamides; Crystallography, X-Ray; Escherichia coli; Free Radicals; Hydrogen; Klebsiella; Ligands; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Molecular Structure; Organometallic Compounds; Photochemistry; Propanediol Dehydratase; Protein Binding; Protein Conformation; Recombinant Fusion Proteins; Vitamin B 12