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

Background: Adenosylcobalamin (coenzyme B-12) serves as a cofactor for enzy matic radical reactions. The adenosyl radical, a catalytic radical in these reactions, is formed by homolysis of the cobalt-carbon bond of the coenzym e, although the mechanism of cleavage of its organometallic bond remains un solved. Results: We determined the three-dimensional structures of diet dehydratase complexed with adeninylpentylcobalamin and with cyanocobalamin at 1.7 Angs trom and 1.9 Angstrom 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 c oenzyme, 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 am ide nitrogen atoms, a sidechain hydroxyl group, and a water molecule. As co mpared with the cyanocobalamin complex, the sidechain of Ser alpha 224 rota tes by 120 degrees to hydrogen bond with N(3) of the adenine ring. Conclusions: The structure of the adenine-ring-binding site provides a mole cular 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-carb on bond. Rotation of the ribose moiety around the glycosidic linkage makes the 5'-carbon radical accessible to the hydrogen atom of the substrate to b e abstracted.