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

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.

Chemical investigations of male and female leaf extracts from Schinus molle L.

The pepper-tree Schinus molle is an evergreen ornamental plant with various and diversified list of medical uses. In this article we analysed the chemical composition of male and female leaves of this plant during the off-flowering and flowering seasons. The leaf extracts were obtained by using a sequential extraction with solvents of different polarities and the chemical composition was investigated by GC-MS. The results showed a total of twenty-three components, in which elemol is the most abundant constituent followed by bicyclogermacrene, γ-eudesmol, α-eudesmol, ß-eudesmol and isocalamendiol. The petroleum ether and diethyl ether extracts from male and female flowering and off-flowering leaves consisted of sesquiterpene hydrocarbons as a major constituent followed by monoterpene hydrocarbons, while the acetone extracts showed a different composition. The obtained results show differences in the chemical composition between male and female and flowering and not flowering.

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

A variety of strategies is applied to alleviate thermodynamic and kinetic limitations in biocatalytic carboxylation of metabolites in vivo. A key feature to consider in enzymatic carboxylations is the nature of the cosubstrate: CO2 or its hydrated form, bicarbonate. The substrate binding and activation mechanism determine what the actual carboxylation agent is. Dihydroxybenzoic acid (de)carboxylases catalyze the reversible regio-selective ortho-(de)carboxylation of phenolics. These enzymes have attracted considerable attention in the last 10 years due to their potential in substituting harsh conditions typical of chemical carboxylations (100-200 °C, 5-100 bar) with, ideally, greener ones (20-40 °C, 1 bar). They are reported to use bicarbonate as substrate, needed in large excess to overcome thermodynamic and kinetic limitations. Therefore, CO2 can be used as substrate by these enzymes only if it is converted into bicarbonate in situ. In this contribution, we report the simultaneous amine-mediated conversion of CO2 into bicarbonate and the ortho-carboxylation of different phenolic molecules catalyzed by 2,3-dihydroxybenzoic acid (de)carboxylase from Aspergillus oryzae. Our results show that under the newly developed conditions a significant thermodynamic (up to twofold increase in conversion) and kinetic improvement (up to approx. fivefold increase in rate) of the biocatalytic carboxylation of catechol is achieved.

Biocatalytic carboxylation of phenol derivatives: kinetics and thermodynamics of the biological Kolbe-Schmitt synthesis.

Microbial decarboxylases, which catalyse the reversible regioselective ortho-carboxylation of phenolic derivatives in anaerobic detoxification pathways, have been studied for their reverse carboxylation activities on electron-rich aromatic substrates. Ortho-hydroxybenzoic acids are important building blocks in the chemical and pharmaceutical industries and are currently produced via the Kolbe-Schmitt process, which requires elevated pressures and temperatures (≥ 5 bar, ≥ 100 °C) and often shows incomplete regioselectivities. In order to resolve bottlenecks in view of preparative-scale applications, we studied the kinetic parameters for 2,6-dihydroxybenzoic acid decarboxylase from Rhizobium sp. in the carboxylation- and decarboxylation-direction using 1,2-dihydroxybenzene (catechol) as starting material. The catalytic properties (K(m), V(max)) are correlated with the overall thermodynamic equilibrium via the Haldane equation, according to a reversible random bi-uni mechanism. The model was subsequently verified by comparing experimental results with simulations. This study provides insights into the catalytic behaviour of a nonoxidative aromatic decarboxylase and reveals key limitations (e.g. substrate oxidation, CO2 pressure, enzyme deactivation, low turnover frequency) in view of the employment of this system as a 'green' alternative to the Kolbe-Schmitt processes.