Substrate-induced conformational change of a coenzyme B12-dependent enzyme:: Crystal structure of the substrate-free form of diol dehydratase

Substrate binding triggers catalytic radical formation through the cobalt-carbon bond homolysis in coenzyme B-12-dependent enzymes. We have determined the crystal structure of the substrate-free form of Klebsiella oxytoca diol dehydratase(.)cyanocobalamin complex at 1.85 Angstrom resolution. The structure contains two units of the heterotrimer consisting of alpha, beta, and gamma subunits. As compared with the structure of its substrate-bound form, the beta subunits are tilted by similar to3degrees and cobalamin is also tilted so that pyrrole rings A and D are significantly lifted up toward the substrate-binding site, whereas pyrrole rings B and C are only slightly lifted up. The structure revealed that the potassium ion in the substrate-binding site of the substrate-free enzyme is also heptacoordinated; that is, two oxygen atoms of two water molecules coordinate to it instead of the substrate hydroxyls. A modeling study in which the structures of both the cobalamin moiety and the adenine ring of the coenzyme were superimposed onto those of the enzyme-bound cyanocobalamin and the adenine ring-binding pocket, respectively, demonstrated that the distortions of the Co-C bond in the substrate-free form are already marked but slightly smaller than those in the substrate-bound form. It was thus strongly suggested that the Co-C bond becomes largely activated (labilized) when the coenzyme binds to the apoenzyme even in the absence of substrate and undergoes homolysis through the substrate-induced conformational changes of the enzyme. Kinetic coupling of Co-C bond homolysis with hydrogen abstraction from the substrate shifts the equilibrium to dissociation.