How a protein generates a catalytic radical from coenzyme B 12: X-ray structure of a diol-dehydratase–adeninylpentylcobalamin complex

Abstract

Background: 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. Results: We determined the three-dimensional structures of diol dehydratase complexed with adeninylpentylcobalamin and with cyanocobalamin at 1.7 Å and 1.9 Å 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 Serα224 rotates by 120° to hydrogen bond with N(3) of the adenine ring. Conclusions: 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.