Computational Study of Glycerol Binding within the Active Site of Coenzyme B₁₂-Dependent Diol Dehydratase

Molecular dynamics (MD) simulations have been employed for the first time to gain insight into the geometry of glycerol (GOL) bound within the active site of B₁₂-dependent diol dehydratase (B₁₂-dDDH). A peculiar feature of the B₁₂-dDDH enzyme is that it undergoes suicidal inactivation by the substrate glycerol. To fully understand the inactivation mechanism, it is crucial to identify all possible interactions between GOL and the surrounding amino acid residues in the enzyme-substrate complex. Particularly important is the orientation of the C3-OH group in GOL since the presence of this OH group is the only difference between GOL and propanediol (PDO), a substrate for B₁₂-dDDH that does not induce suicidal inactivation. The MD simulations indicate that glycerol can adopt two conformations that differ with respect to the orientation of the C3-OH group; in one conformer, the C3-OH group is oriented toward Ser301 (C3-OH···Ser301), and in the other toward Asp335 (C3-OH···Asp335). Although the former configuration is consistent with the crystal structure of B₁₂-dDDH crystallized with cyanocobalamin (CNCbl) as the cofactor, MD simulations of this system suggest a substantial predominance of the latter conformer. A similar result with an even higher preference for the latter conformer is obtained for B₁₂-dDDH with 5'-deoxyadenosylcobalamin (AdoCbl) as a cofactor. Employing QM/MM calculations it is found that the energy difference between the two conformers of GOL is very small in CNCbl B₁₂-dDDH, where the slightly preferred conformer is C3-OH···Ser301. However, in AdoCbl B₁₂-dDDH, this energy difference is higher, implying that GOL exists predominantly as the C3-OH···Asp335 conformer. These findings offer a new perspective for investigations of substrate-induced inactivation of the B₁₂-dDDH enzyme.