The crystal structure of coenzyme B₁₂-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably α₂β₂γ₂. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an iso-functional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (K_m(R)/K_m(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 Å resolution. The enzyme exists as a dimer of the αβγ heterotrimer. Cobalamin is bound at the interface between the α and β subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (β/α)₈ barrel that was formed by a central region of the α subunit. The substrate propane-1,2-diol and essential cofactor K⁺ are bound inside the (β/α)₈ barrel above the corrin ring of cobalamin. K⁺ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the α and β subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.