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

Abstract

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 B 12 -dependent diol dehydratase (B 12 -dDDH). A peculiar feature of the B 12 -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 B 12 -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 B 12 -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 B 12 -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 B 12 -dDDH, where the slightly preferred conformer is C3-OH••• Ser301. However, in AdoCbl B 12 -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 B 12 -dDDH enzyme.

Recent Progress in the Understanding and Engineering of Coenzyme B12-Dependent Glycerol Dehydratase

“…Therefore, engineering an enzyme that is resistant to inactivation should be an important research direction in applying coenzyme B 12 -dependent GDHts for industrial processes. Structural and computational studies have demonstrated that the conformation of glycerol plays a role in GDHt inactivation (Bachovchin et al, 1977;Bilicì et al, 2019). Mutations were introduced at the active site of coenzyme B 12 -dependent DDHt to favor its interaction with the pro-R conformation, and the variants showed slower inactivation rates than the wild-type enzyme even though their activities decreased (Yamanishi et al, 2012).…”

“…In another computational study, Biliæ et al also reported the interaction of the two conformations of glycerol at the active site of Kp DDHt with respect to the orientation of the C3-OH group. The OH group in the pro- S conformation was oriented toward Ser301 of the α-subunit, and that in the pro- R conformation was oriented toward Asp335 ( Figures 7C,D ; Bilicì et al, 2019 ). An attempt was made to introduce mutations into coenzyme B 12 -dependent DDHt to favor its interaction with the pro- R conformation of glycerol ( Yamanishi et al, 2012 ), which will be described later.…”

“…(A,B) Hydrogen abstracted in the first catalytic step are shown by arrows. (C,D) The C3-OH group of glycerol is oriented toward Ser301 in the pro- S conformation while toward Asp335 in the pro- R conformation (Modified with permission from Bilicì et al, 2019 ).…”

“…(B) The mechanism of reactivation via cofactor exchange by GDHt reactivase. E: apoenzyme; AdoCbl: adenosylcobalamin; S: substrate; P: product; RF: reactivase; AdoH: 5 -deoxyadenosine; Cbl: cobalamin or damaged cofactor (Modified with permission fromBilicì et al, 2019).…”

Glycerol as a Substrate and Inactivator of Coenzyme B12‐Dependent Diol Dehydratase

“…This observation led to our subsequent classical MD simulation study which detailed the binding geometries of pro(R)-and pro (S)-GOL and (R)-and (S)-1,2-PDO in B 12 -dDDH with AdoCbl and CNCbl as the cofactors. [26] In all model systems containing the Figure 1. Accepted mechanism of B 12 -dDDH glycerol catalysis (green arrows) and the inactivation mechanism proposed by Doitomi et al [17] (red arrows).…”