Theoretical study of the reaction mechanism of phenolic acid decarboxylase

Abstract

The cofactor‐free phenolic acid decarboxylases (PADs) catalyze the non‐oxidative decarboxylation of phenolic acids to their corresponding p‐vinyl derivatives. Phenolic acids are toxic to some organisms, and a number of them have evolved the ability to transform these compounds, including PAD‐catalyzed reactions. Since the vinyl derivative products can be used as polymer precursors and are also of interest in the food‐processing industry, PADs might have potential applications as biocatalysts. We have investigated the detailed reaction mechanism of PAD from Bacillus subtilis using quantum chemical methodology. A number of different mechanistic scenarios have been considered and evaluated on the basis of their energy profiles. The calculations support a mechanism in which a quinone methide intermediate is formed by protonation of the substrate double bond, followed by C–C bond cleavage. A different substrate orientation in the active site is suggested compared to the literature proposal. This suggestion is analogous to other enzymes with p‐hydroxylated aromatic compounds as substrates, such as hydroxycinnamoyl‐CoA hydratase‐lyase and vanillyl alcohol oxidase. Furthermore, on the basis of the calculations, a different active site residue compared to previous proposals is suggested to act as the general acid in the reaction. The mechanism put forward here is consistent with the available mutagenesis experiments and the calculated energy barrier is in agreement with measured rate constants. The detailed mechanistic understanding developed here might be extended to other members of the family of PAD‐type enzymes. It could also be useful to rationalize the recently developed alternative promiscuous reactivities of these enzymes.

Regioselective para‐Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase

“…This intermediate has chemical similarity to that proposed for the phenolic acid decarboxylases (PAD)8a and is different from the 1,3‐dipolar cycloaddition mechanism proposed for Fdc1 14a, 16, 17, 18. The calculations, employing a large model of the active site with 283 atoms (Figure 2 b), suggested that the generation of a cycloadduct is unlikely in the case of AroY as it would require the formation of a very strained intermediate (Figure S29).…”

Application of quantum mechanics/molecular mechanics methods in the study of enzymatic reaction mechanisms

“…The cluster modeling approach has provided valuable information for many enzymatic reactions, and has been used with success by many research groups including ourselves through the past decades, and is particularly efficient in discriminating between different mechanistic proposals and in studying metalloenzymes . While historically, this approach has started with systems as small as 20–30 atoms, present applications have been using much larger atomistic regions comprising up to 300–400 atoms . Despite the pragmatic nature of the cluster modeling approach and large number of enzymes successfully studied with this method, there are several cases for which its application has been problematic, or even impossible.…”

Biotransformation of Plant-Derived Phenolic Acids

“…[61][62][63] These decarboxylases have been identified in several bacteria and fungi. [68] The enzyme can convert a wide variety of hydroxycinnamic acids including pCA, CA, FA, and SiA to their corresponding 4-vinyl derivatives (Figure 2). Based on theoretical studies, this enzyme catalyzes non-oxidative decarboxylation via formation of a quinone methide intermediate followed by C-C bond cleavage.…”