A Theoretical Study of the Benzoylformate Decarboxylase Reaction Mechanism

Abstract

Density functional theory calculations are used to investigate the detailed reaction mechanism of benzoylformate decarboxylase, a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the nonoxidative decarboxylation of benzoylformate yielding benzaldehyde and carbon dioxide. A large model of the active site is constructed on the basis of the X-ray structure, and it is used to characterize the involved intermediates and transition states and evaluate their energies. There is generally good agreement between the calculations and available experimental data. The roles of the various active site residues are discussed and the results are compared to mutagenesis experiments. Importantly, the calculations identify off-cycle intermediate species of the ThDP cofactor that can have implications on the kinetics of the reaction.

Computational characterization of enzyme-bound thiamin diphosphate reveals a surprisingly stable tricyclic state: implications for catalysis

“…Models B – E represent the cofactor in the BFDC active site in the absence and presence of bound ligands. The active site model is built on the basis of the crystal structure (PDB ID 1MCZ) and is identical to that used in the mechanistic study [29]. As shown in Fig.…”

“…Subsequently, a similar approach was used to characterize the nucleophilicity of the N1′ and N4′ centers [24]. In many cases, rather than simply focus on the cofactor, computational studies have been used to investigate full reaction mechanisms of ThDP enzymes, including pyruvate decarboxylase (PDC) [25–28], benzoylformate decarboxylase (BFDC) [29–30], acetohydroxy acid synthase [24,31–35], pyruvate dehydrogenase (PDH) [36], benzaldehyde lyase [37], cyclohexane dione hydrolase [38], oxalyl-CoA decarboxylase [39], DXP synthase [40] and transketolase [41–42].…”

“…In a very recent study, we used quantum chemical methodology to investigate the detailed reaction mechanism of benzoylformate decarboxylase (BFDC) [29]. A model of the active site was designed on the basis of the X-ray structure of BFDC in complex with the substrate analog inhibitor, ( R )-mandelate.…”

“…It was found to be about 5 kcal/mol lower in energy than the IP state, thereby raising the barrier for the formation of the cofactor–substrate adduct (C2α-mandelyl–ThDP). Of course this has important implications for the overall kinetics of any BFDC-catalyzed reaction and, potentially, for all THDP-dependent enzymes [29].…”

Characterization of Thiamine Diphosphate-Dependent 4-Hydroxybenzoylformate Decarboxylase Enzymes from Rhodococcus jostii RHA1 and Pseudomonas fluorescens Pf-5 Involved in Degradation of Aryl C2 Lignin Degradation Fragments

“…Further mutational studies showed that the effect of replacement of His-281 or His-70 on catalysis is dependent on the amino acid substituted (40). Further work using density functional theory calculations highlighted the probability that His-70, 281 and Ser-26 are all involved in substrate binding via hydrogen bonding (41).…”