Quantum Chemistry as a Tool in Asymmetric Biocatalysis: Limonene Epoxide Hydrolase Test Case

Abstract

Quantum chemical models of enzyme active sites have in recent years proven to be a very powerful tool in the elucidation of enzymatic reaction mechanisms. [1] In the socalled cluster approach, a limited part of the enzyme around the active site is cut out and treated using relatively accurate electronic structure methods, typically hybrid density functional theory (DFT). The missing enzyme surrounding is approximated by a homogeneous polarizable continuum model with some assumed dielectric constant. [2] A large variety of enzymatic systems have been investigated quite successfully using this approach and a wealth of mechanistic insight has been gained. [3] A few years ago, active site models consisted typically of less than 100 atoms. However, with today s computers and using the same computational protocol, it is possible to treat more than 250 atoms quite routinely. These developments have paved the way for wider applications of the cluster approach, beyond the pure mechanistic investigations.To investigate and explain sources of various kinds of selectivities, in particular enantioselectivity, one has typically to reproduce relative transition-state energies on the order of 1 kcal mol À1 . The accuracy of modern DFT methods has been proven to be sufficiently high to achieve this. In particular, these methods have in recent years been applied very successfully to a multitude of problems in the field of asymmetric homogenous catalysis. [4] Modeling enzymatic enantioselectivity with the cluster approach has remained somewhat out of reach because larger active-site models are in general required to create the chiral environment provided by the enzyme. Herein, we will demonstrate that this kind of modeling approach is indeed able to reproduce and rationalize enantioselectivity in enzymes and has thus the potential to become a valuable tool also in the field of asymmetric biocatalysis.Today enzymes are increasingly used in synthetic chemistry for the production of base and fine chemicals. [5] Indeed, biocatalytic processes are starting to replace transition-metalcatalyzed reactions for large-scale production of drug compounds. [6] One of the most attractive features of enzymes in this respect is their high levels of selectivity. Various engineering techniques have been developed to manipulate the properties of enzymes to achieve the desired function. These techniques range from pure combinatorial approaches to semi-rational and fully rational designs, which are based on the detailed knowledge of the structure and mechanism of the enzymes. Theoretical methodology in asymmetric biocatalysis has relied mainly on substrate docking or molecular dynamics simulations of the enzyme-substrate (ES) complexes in the ground state. [7,8] To some extent, this has been successful in qualitatively guiding the experimental work as to which parts of the active site to manipulate and also to provide some rationalization for observed trends. These approaches are, however, inherently deficient since they do not consider the transition ...

Understanding Complex Mechanisms of Enzyme Reactivity: The Case of Limonene-1,2-Epoxide Hydrolases

“…Our results show that LEH exploits a concerted mechanism to catalyze epoxide opening; corroborating previous findings, 30,31 we have shown that LEH regioselectivity is guided by two different transition states: the preferred attack on the most substituted carbon atom is mediated by a chair-like LEO conformation, while a less stable twist-boat conformation leads the attack on the less substituted carbon atom. In addition, the (ligand dependent) organization of the active site results in a different first event of the catalytic activation, and this in turn defines the regiochemical outcome of the hydrolysis reaction.…”

“…Indeed, several studies have already demonstrated the crucial role of monomer-monomer interactions in stabilizing and tuning LEH dynamics and stability. 23,25,47 Therefore, unlike previous computational characterizations, 28,[30][31][32] the whole dimer has been considered in our simulations and consequentially the different substrates have been modeled in both monomeric chains (Figure 1a). In addition to improving the reliability of the model, this allows increased sampling of the conformational landscape of the catalytic pockets.…”

“…The Himo group used a DFT cluster approach to build a simplified LEH active site model (i.e., only some key residues around the catalytic pocket were explicitly considered) that shed light on the enzyme catalytic mechanism. 30,31 These studies demonstrated that theoretical methodologies that provide a quantum mechanical characterization of transition states can generate a quantitative description of catalytic mechanisms and can be also used as tools in the field of asymmetric biocatalysis. 31 According to Himo et al's results, LEH reacts by a one-step SN2-like general acid/general base-catalyzed mechanism that significantly differs from the two-step general basecatalyzed reaction of the other EH family members; this is supported by a hybrid quantum mechanics/molecular mechanics (QM/MM) study 32 .…”

“…30,31 These studies demonstrated that theoretical methodologies that provide a quantum mechanical characterization of transition states can generate a quantitative description of catalytic mechanisms and can be also used as tools in the field of asymmetric biocatalysis. 31 According to Himo et al's results, LEH reacts by a one-step SN2-like general acid/general base-catalyzed mechanism that significantly differs from the two-step general basecatalyzed reaction of the other EH family members; this is supported by a hybrid quantum mechanics/molecular mechanics (QM/MM) study 32 . In particular, the Asp101-Arg99-Asp132 catalytic triad appears to drive a concerted reaction in which Asp132 acts as a general base, extracting a proton from a water molecule.…”

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

“…114,117,118 In many cases, however, we have found that B3LYP still remains a safe choice, particularly when used in combination with large basis sets and with a dispersion correction. 51,[119][120][121][122] The errors of B3LYP are quite systematic (underestimation of barriers by~3 kcal mol −1 ) which enables its conscientious use.…”