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Diol dehydratase, dependent on coenzyme B12 (B12 -dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B12 -independent glycerol dehydratase (B12 -iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B12 -dDDH and that of resistance of B12 -iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B12 -dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B12 -iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.

Računalne simulacije molekulske dinamike, QM i QM/MM računi upućuju da je inaktivacija djelomično posljedica velike pokretljivosti kofaktora unutar enzima. Upravo iz tog razloga kofaktor može započeti kemijsku pretvorbu substrata glicerola na dva kemijski ekvivalentna kraja. Kemijska pretvorba na jednom kraju glicerola vodi u katalizu, kemijska pretvorba na drugom kraju vodi u inaktivaciju.

Microbial conversion of crude glycerol, the waste from biofuel production, into compounds of greater industrial value could solve technical difficulties encountered by the conventional means of chemical conversion [1]. During the particular type of microbial conversion, in the first step glycerol undergoes dehydration by enzymes dehydratases into 3‐hydroxylpropionaldehyde (3HPA) [2, 3]. Two classes of dehydratases can catalyze dehydration of glycerol, B12‐independent and B12‐dependent dehydratases, from which B12‐ dependent class is more often used due to its tolerance to aerobic conditions [4]. However, a peculiar property of B12‐dependent dehydratases is that glycerol, which is their substrate, also acts as an irreversible inhibitor [5]. Based on the B12‐dependent diol dehydratase (B12‐dDDH) crystal structure with glycerol (PDB code: 3AUJ), K. Yoshizawa et al. concluded that the geometry of such bound glycerol enables radical reorganization thus causing inhibition. [6] However, in the recent study on similar enzyme B12‐dependent glycerol dehydratase we observed glycerol in a different geometry [7]. Here we present a detailed computational study of glycerol binding within the active site of B12‐dDDH and find a binding geometry similar to one observed in Ref. 7 (Figure 1). We consider larger implications of our findings for the mechanism of substrate induced inactivation.

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 (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 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 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.

During biodiesel production a large amount of glycerol is created as a byproduct [1, 2]. Dehydration of glycerol to a more valuable product could be obtained by eco-friendly methods utilizing B12-dependent dehydratases which turn glycerol into 3-hydroxypropanal [3-5]. However, the unusual property of glycerol is that it is both the supstrate and the irreversible inhibitor of B12-dependent dehydratases [6]. The proposed mechanism of inactivation is based on the crystal structure of B12-dependant diol dehydratase (DDH, PDB code: 3AUJ) [7]. This mechanism relies on the position of the hydroxyl group at C(3) atom of glycerol being oriented towards the serine residue (Ser301) in the active site. However, incompleteness of the enzyme in crystal structure (missing adenosyl group), poor resolution (2.1 Å) and small electron densityaroud C(3) call for further research. Herein we compare geometrical parameters of the active site of DDH obtained by molecular dynamic simulations (MD) and quantum chemical calculations (DFT) with the crystal structure, and investigate various patterns ofglycerol binding in the active site.