Evaluation of the Substrate Scope of Benzoic Acid (De)carboxylases According to Chemical and Biochemical Parameters

Abstract

The enzymatic carboxylation of phenolic compounds has been attracting increasing interest in recent years, owing to its regioselectivity and technical potential as a biocatalytic equivalent for the Kolbe-Schmitt reaction. Mechanistically the reaction was demonstrated to occur through electrophilic aromatic substitution/water elimination with bicarbonate as a cosubstrate. The effects of the substituents on the phenolic ring have not yet been elucidated in detail, but this would give detailed insight into the substrate-activity relationship and would provide predictability for the acceptance of future substrates. In this report we show how the kinetic and (apparent) thermodynamic behavior can be explained through the evaluation of linear free energy relationships based on electronic, steric, and geometric parameters and through the consideration of enzyme-ligand interactions. Moreover, the similarity between the benzoic acid decarboxylases and the amidohydrolases superfamily is investigated, and promiscuous hydrolytic activity of the decarboxylase in the context of the hydrolysis of an activated ester bond has been established.

Carboxylation of Hydroxyaromatic Compounds with HCO3− by Enzyme Catalysis: Recent Advances Open the Perspective for Valorization of Lignin-Derived Aromatics

“…Linear free-energy relationships (LFERs) analysis of the substrate scope of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae was used for mechanism investigation [76]. Although the reaction mechanism of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae is not known, its substrate scope is considerably larger if compared to the limited range of substrates carboxylated by 2,6-dihydroxybenzoic acid decarboxylase from Rhizobium sporomusa.…”

“…Therefore 2,3-DHBD_Ao was considered suitable for an LFERs study. Twelve different di-substitited aromatic substrates with only one OH-substituent (except for catechol) were carboxylated by the enzyme and the paraor meta-effect of the X-substituent was referred to the carboxylic directing group (Figure 18) [76].…”

“…Therefore 2,3-DHBD_Ao was considered suitable for an LFERs study. Twelve different di-substitited aromatic substrates with only one OH-substituent (except for catechol) were carboxylated by the enzyme and the para-or meta-effect of the X-substituent was referred to the carboxylic directing group (Figure 18) [76]. The carboxylation reactions were carried out in HPLC screw-capped glass vials with E. coli whole lyophilized cells transformed with the 2,3-DHBD_Ao enzyme according to the following reaction conditions: 10 mM substrate, 30 mg/mL whole cells expressing 2,3-dihydroxybenzoic acid Figure 18.…”

“…Correlation of the logarithm of the calculated initial reaction rates (v) with respect to the Taft constants (σ 0 ) for the selected set of substrates shown inFigure 18, (R 2 = 0.92). Reprinted with permission from[76]. Copyright 2016, John Wiley and Sons.…”

Amine‐Mediated Enzymatic Carboxylation of Phenols Using CO2 as Substrate Increases Equilibrium Conversions and Reaction Rates

“…and a K M value for bicarbonate in the molar concentration range, it is reasonable to assume that the decarboxylation of ortho ‐hydroxybenzoic acids (salicylic acids) is the physiologically relevant reaction. Crystallographic/mechanistic data and structure‐activity relationships revealed that the carboxylation reaction occurs by electrophilic aromatic substitution, where an essential Zn 2+ cation coordinated in the active site of the enzyme behaves as a Lewis acid, activating bicarbonate for the nucleophilic attack exerted by the ortho ‐carbon of the phenolic substrate, which itself is activated by an aspartate residue. This reaction has attracted considerable attention because of the resemblance to the Kolbe–Schmitt synthesis of salicylic acids, which contrary to the biocatalytic variant requires harsh reaction conditions (100–200 °C, 5–100 bar) and suffers from regioselectivity issues.…”