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

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 A 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 approximately 3 degrees 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.

DFT Analysis of Co−Alkyl and Co−Adenosyl Vibrational Modes in B12-Cofactors

“…Recent crystallographic studies for two B 12 -containg enzymes, glutamate mutase (GLM) 48 and diol dehydratase (DDH), 49 suggest that the main contribution to Co-C bond cleavage is substrate-induced deformation of the coenzyme, although the evidence is complicated by the likelihood that the Co(III) is reduced to Co(II) in the crystals. Recent QM/MM calculations reported by Jensen and Ryde 50 likewise support adenosyl deformation as the most feasible mechanism of Co-C activation.…”

Crystal Structures of Ethanolamine Ammonia-lyase Complexed with Coenzyme B12 Analogs and Substrates

“…Substrate Switch-The structures of two AdoCbl-dependent enzymes, methylmalonyl-CoA mutase and diol dehydratase, reveal that substrate binding causes significant conformational changes at the overall fold level (50,51). The bound substrate pushes Gln␣ 336 and Phe␣ 374 toward pyrrole rings A and D of the corrin ring in diol dehydratase.…”

“…As a result, this enzyme displays movement of cobalamin and the ␤-subunit with respect to the ␣-subunit upon substrate binding, which causes the tilting of cobalamin. This is considered responsible for the substrate switch or substrate-induced additional labilization of the Co-C bond (51). To test this possibility, the structure of the substrate-free form of EAL was also analyzed.…”

“…The Co-C bond in the substrate-free form of diol dehydratase is also distorted and already activated. Substrate binding increases its steric distortion, which is an entity of the substrate-induced additional labilization of the Co-C bond (51). Apparent substrate triggering of the Co-C bond cleavage would be due to the shift of equilibrium toward the formation of much more stable substrate radical.…”

Experimental Design For 1,3-Propanediol Biosynthesis by K. Pneumoniae GLC29 Using Glycerol

“…The addition from 5 mg/l to 20 mg/l of vitamin B12, production was negatively impacted because of its interaction with glycerol. An irreversible binding of the vitamin B12 and glycerol with the enzyme, forms alkylcobalamines, and to avoid low activity of the enzyme, the amount of glycerol should be controlled [20,[29][30][31][32], but also the amount of vitamin should be controlled. As a consequence of the normal catalytic cycle with glycerol, the coenzyme B12 is occasionally rendered inactive (B12-inact).…”