The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme‐catalyzed para‐carboxylation of catechols, employing 3,4‐dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN‐dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN‐associated 1,3‐dipolar cycloadditions in related enzymes.

The Pictet–Spengler (PS) reaction, i.e., the acid-catalyzed condensation between β-arylethylamine and an aldehyde or a ketone and the subsequent ring closure, is an important reaction in organic chemistry. A number of enzymes (called Pictet–Spenglerases, PSases) have been identified to catalyze this reaction, usually with very high enantioselectivity, making these enzymes of potential value in biocatalysis. PSases catalyze the key step in the biosynthesis of indole and benzylisoquinoline alkaloids of plant origin, some of which have pharmacological importance. However, the reaction mechanisms and the origins of the selectivity are not fully understood. Herein, we report a quantum chemical investigation of the mechanism and enantioselectivity of norcoclaurine synthase (NCS), an enzyme that catalyzes the PS condensation between dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA). A large model of the active site is designed on the basis of a recent crystal structure, and it is used to calculate the detailed energy profile of the reaction. Good agreement is obtained between the calculated energies and available experimental information. Both the “dopamine-first” and the “HPAA-first” binding modes of the substrates reported in the literature are shown to be energetically accessible in the enzyme–substrate complex. However, it is demonstrated that only the dopamine-first pathway is associated with feasible energy barriers. Key active site residues are identified, and their roles in the catalysis are discussed and compared to site-directed mutagenesis experiments. Very importantly, the calculations are able to reproduce and rationalize the observed enantioselectivity of NCS. A detailed analysis of the geometries of the intermediates and transition states helps to pinpoint the main factors controlling the selectivity.

The computational study of enantioselective reactions is a challenging task that requires high accuracy, as very small energy differences have to be reproduced. Quantum chemical methods, most commonly density functional theory, are today an important tool in this pursuit. This Perspective describes recent efforts in modeling asymmetric reactions in enzymes by means of the quantum chemical cluster approach. The methodology is described briefly and a number of illustrative case studies performed recently at our laboratory are presented. The reviewed enzymes are limonene epoxide hydrolase, soluble epoxide hydrolase, arylmalonate decarboxylase, phenolic acid decarboxylase, benzoylformate decarboxylase, secondary alcohol dehydrogenase, acyl transferase, and norcoclaurine synthase. The challenges encountered in each example are discussed, and the modeling lessons learned are highlighted.

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.

The cofactor-free phenolic acid decarboxylases (PADs) catalyze the nonoxidative decarboxylation of phenolic acids to their corresponding p-vinyl derivatives. Since these compounds are useful industrially, PADs have potential applications as biocatalysts. Recently, PADs have been reported to also catalyze the hydration and carboxylation of hydroxystyrenes, increasing further their biocatalytic utility. We have used quantum chemical methodology to investigate the detailed mechanisms of both promiscuous reactions. A large model of the active site is designed starting from the crystal structure of PAD from Bacillus subtilis. The calculations suggest new mechanisms, quite different from the literature proposals. For the carboxylation reaction, a carbon dioxide molecule is proposed to be generated from bicarbonate first and then act as the source for the carboxylate group of the product. For the hydration activity, the reaction is suggested to start with the formation of a quinone methide intermediate by protonation of the CC double bond of the p-vinylphenol substrate. A water molecule then attacks the α-carbon to generate the alcohol product. The enantioselectivity of the hydration reaction is also investigated in this study, and the calculations are able to reproduce and rationalize the observed experimental outcome.

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from Polaromonas sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of γ-RSD from Rhizobium sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from Rhizobium sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct.

Strictosidine synthase (STR) catalyzes the Pictet–Spengler (PS) condensation between tryptamine and secologanin, leading to the formation of (S)-strictosidine. The product is a central intermediate in the biosynthesis of various indole alkaloids, many of which are pharmaceutically important. STRs have relatively broad scopes for both the amine and aldehyde substrates, making them attractive biocatalyst candidates for applications in organic synthesis. An interesting feature of STR discovered recently is the switched enantiopreference of the reaction using short-chain aliphatic aldehydes in comparison to the natural secologanin substrate. Herein, we use quantum chemical calculations to investigate the detailed reaction mechanism and the origins of enantioselectivity of STR, considering both natural and non-natural substrates. The calculated overall barrier is in a good agreement with the measured rate constant, and the nature of the rate-determining step is consistent with kinetic isotope effect experiments. Importantly, the enantioselectivity for the reactions of both natural and non-natural substrates are well reproduced. A systematic analysis of energy profiles of the entire reactions and the geometries of the key transition states and intermediates along the pathways leading to the different enantiomers of the products provides a rationalization for the observed inversion of enantioselectivity.

The acyl transferase from Mycobacterium smegmatis (MsAcT) catalyzes the acyl transfer between a range of primary and secondary alcohols, whereby its outstanding ability is to perform this reaction in aqueous solution. Therefore, MsAcT opens different options for acylation reactions enabling alternatives for many conventionally hydrolytic enzymes used in biocatalysis. Nevertheless, hydrolysis is still a major side reaction of this enzyme. To provide a detailed understanding of the competition between hydrolysis and transesterification reactions, a combination of density functional theory and free energy perturbation methods have been employed. The relative binding free energies and the energy profiles of the chemical steps involved in the reaction were calculated for a number of substrates. The calculations show that the enzyme active site exhibits a higher affinity for substrates with an aromatic ring. The rate-determining step corresponds to the collapse of a negatively charged tetrahedral intermediate in the substrate acylation half-reaction. The intrinsic barriers of the transesterification and hydrolysis half-reactions are calculated to be of similar heights, suggesting that the determining factor in the MsAcT specificity is the higher binding affinity of the active site for the alcohol substrates relative to water. Finally, the influence of the acyl donor on the MsAcT-catalyzed reaction is also investigated by considering different esters in the calculations.