Computational evaluation of zirconocene catalysts for ε-caprolactone cationic ring-opening polymerization.

This quantum chemical study presents the ligand effect and a structure-property relationship in the cationic ring-opening polymerization (CROP) of ε-caprolactone using zirconocene catalysts. We first examined the effects of catalyst structure on the initiation and chain propagation steps of the CROP process. A total of 54 catalyst structures were investigated to understand the influence of the ligand structure on the stability of the catalyst-monomer complex and polymerization activity. The properties of the catalysts were analyzed in terms of ancillary ligands, ligand substituents, and bridging units. Calculations showed that the polymerization follows a proposed cationic mechanism, with ring opening occurring via alkyl-bond cleavage. A correlation between complex stability and activation energy was also observed, with ligand substituents dominating in both steps. While the ancillary ligands directly affect the HOMO energy level, the bridges are mainly responsible for the catalyst geometries, resulting in reduced complex stability and higher activation energy for the propagation step. This study contributes to a better understanding of the structural characteristics of zirconocene catalysts, which offers guidance for improving CROP activities in lactone polymerization.

Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1-OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1.

Arabidopsis thaliana cell-wall invertase 1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the -1 furanosyl ring underwent a conformational change corresponding to 3E ↔ [E2]⧧ ↔ 1E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH···nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.

A computational study of the reaction mechanism and stereospecificity of dihydropyrimidinase.

Dihydropyrimidinase (DHPase) is a key enzyme in the pyrimidine pathway, the catabolic route for synthesis of ß-amino acids. It catalyses the reversible conversion of 5,6-dihydrouracil (DHU) or 5,6-dihydrothymine (DHT) to the corresponding N-carbamoyl-ß-amino acids. This enzyme has the potential to be used as a tool in the production of ß-amino acids. Here, the reaction mechanism and origin of stereospecificity of DHPases from Saccharomyces kluyveri and Sinorhizobium meliloti CECT4114 were investigated and compared using a quantum mechanical cluster approach based on density functional theory. Two models of the enzyme active site were designed from the X-ray crystal structure of the native enzyme: a small cluster to characterize the mechanism and the stationary points and a large model to probe the stereospecificity and the role of stereo-gate-loop (SGL) residues. It is shown that a hydroxide ion first performs a nucleophilic attack on the substrate, followed by the abstraction of a proton by Asp358, which occurs concertedly with protonation of the ring nitrogen by the same residue. For the DHT substrate, the enzyme displays a preference for the L-configuration, in good agreement with experimental observation. Comparison of the reaction energetics of the two models reveals the importance of SGL residues in the stereospecificity of catalysis. The role of the conserved Tyr172 residue in transition-state stabilization is confirmed as the Tyr172Phe mutation increases the activation barrier of the reaction by ∼8 kcal mol-1. A detailed understanding of the catalytic mechanism of the enzyme could offer insight for engineering in order to enhance its activity and substrate scope.

Active site dynamics and catalytic mechanism in arabinan hydrolysis catalyzed by GH43 endo-arabinanase from QM/MM molecular dynamics simulation and potential energy surface.

The endo-1,5-α-L-arabinanases, belonging to glycoside hydrolase family 43 (GH43), catalyse the hydrolysis of α-1,5-arabinofuranosidic bonds in arabinose-containing polysaccharides. These enzymes are proposed targets for industrial and medical applications. Here, molecular dynamics (MD), potential energy surface and free energy (potential of mean force) simulations are undertaken using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to understand the active site dynamics, catalytic mechanism and the electrostatic influence of active site residues of the GH43 endo-arabinanase from G. stearothermophilus. The calculated results give support to the single-displacement mechanism proposed for the inverting GH43 enzymes: first a proton is transferred from the general acid E201 to the substrate, followed by a nucleophilic attack by water, activated by the general base D27, on the anomer carbon. A conformational change (2E ↔E3 ↔ 4E) in the -1 sugar ring is observed involving a transition state featuring an oxocarbenium ion character. Residues D87, K106, H271 are highlighted as potential targets for future mutation experiments in order to increase the efficiency of the reaction. To our knowledge, this is the first QM/MM study providing molecular insights into the glycosidic bond hydrolysis of a furanoside substrate by an inverting GH in solution.Communicated by Ramaswamy H. Sarma.