Substrate-Induced Conformational Change of a Coenzyme B12-Dependent Enzyme: Crystal Structure of the Substrate-Free Form of Diol Dehydratase,

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Abstract
Substrate binding triggers catalytic radical formation through the cobalt−carbon bond homolysis in coenzyme B12-dependent enzymes. We have determined the crystal structure of the substrate-free form of Klebsiella oxytoca diol dehydratase·cyanocobalamin complex at 1.85 Å resolution. The structure contains two units of the heterotrimer consisting of α, β, and γ subunits. As compared with the structure of its substrate-bound form, the β subunits are tilted by ∼3° 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.

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