Alexandra Vardi-Kilshtain

Toward accurate screening in computer-aided enzyme design.

The ability to design effective enzymes is one of the most fundamental challenges in biotechnology and in some respects in biochemistry. In fact, such ability would be one of the most convincing manifestations of a full understanding of the origin of enzyme catalysis. In this work, we explore the reliability of different simulation approaches, in terms of their ability to rank different possible active site constructs. This validation is done by comparing the ability of different approaches to evaluate the catalytic contributions of various residues in chorismate mutase. It is demonstrated that the empirical valence bond (EVB) model can serve as a practical yet accurate tool in the final stages of computer-aided enzyme design (CAED). Other approaches for fast screening are also examined and found to be less accurate and mainly useful for qualitative screening of ionized residues. It is pointed out that accurate ranking of different options for enzyme design cannot be accomplished by approaches that cannot capture the electrostatic preorganization effect. This is in particular true with regard to current design approaches that use gas phase or small cluster calculations and then estimate the interaction between the enzyme and the transition state (TS) model rather than the TS binding free energy or the relevant activation free energy. The ability of the EVB model to provide a tool for quantitative ranking in the final stage of CAED may help in progressing toward the design of enzymes whose catalytic power is closer to that of native enzymes than to that of the current generation of designer enzymes.

Hybrid Quantum and Classical Simulations of the Formate Dehydrogenase Catalyzed Hydride Transfer Reaction on an Accurate Semiempirical Potential Energy Surface.

Formate dehydrogenase (FDH) catalyzes the oxidation of formic acid to carbon dioxide using nicotinamide adenine dinucleotide (NAD(+)) as a cofactor. In the current work we present extensive benchmark calculations for several model reactions in the gas phase that are relevant to the FDH catalyzed hydride transfer. To this end we employ G4MP2 and CBS-QB3 ab initio calculations as well as density functional theory methods. Using these results we develop a specific reaction parameter (SRP) Hamiltonian based on the semiempirical AM1 method. The SRP semiempirical Hamiltonian is subsequently used in hybrid quantum mechanics/molecular mechanics simulations of the FDH catalyzed reaction in Pseudomonas sp. 101 (PseFDH). The classical potential of mean force (PMF) is computed as a function of structural progress coordinates during the course of the hydride transfer reaction: The antisymmetric reactive stretch, the donor-acceptor distance, and an orbital rehybridization coordinate. The quantum PMF is computed using a centroid Feynman path-integral (PI) approach. Subsequently, kinetic isotope effects are computed using a mass-perturbation based PI method. Finally, the antisymmetric stretch vibrational frequency is computed for an azide ion in FDH and in aqueous solution.

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

The Empirical Valence Bond as an Effective Strategy for Computer-Aided Enzyme Design

The ability of the empirical valence bond (EVB) to be used in screening active site residues in enzyme design is explored in a preliminary way. This validation is done by comparing the ability of this approach to evaluate the catalytic contributions of various residues in chorismate mutase. It is demonstrated that the EVB model can serve as an accurate tool in the final stages of computer-aided enzyme design (CAED). The ability of the model to predict quantitatively the catalytic power of enzymes should augment the capacity of current approaches for enzyme design.

Free energy simulations of active-site mutants of dihydrofolate reductase.

This study employs hybrid quantum mechanics-molecular mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue I14 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the I14X mutants (X = V, A, and G) indicated slower hydride transfer rates and increasingly temperature-dependent kinetic isotope effects (KIEs) with systematic reduction of the I14 side chain. The QM/MM simulations show that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier height of the hydride transfer reaction increases relative to the wild-type enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the donor-acceptor distance (DAD) and the active-site hydrogen bonding network compared with those detected in the native enzyme. These observations suggest that the perturbations induced by the mutations partly impair the active-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHFR variants are similar. Crystal structures of I14 mutants (V, A, and G) confirmed the trend of increased flexibility of the M20 and other loops.

Enzyme structure captures four cysteines aligned for disulfide relay.

Thioredoxin superfamily proteins introduce disulfide bonds into substrates, catalyze the removal of disulfides, and operate in electron relays. These functions rely on one or more dithiol/disulfide exchange reactions. The flavoenzyme quiescin sulfhydryl oxidase (QSOX), a catalyst of disulfide bond formation with an interdomain electron transfer step in its catalytic cycle, provides a unique opportunity for exploring the structural environment of enzymatic dithiol/disulfide exchange. Wild‐type Rattus norvegicus QSOX1 (RnQSOX1) was crystallized in a conformation that juxtaposes the two redox‐active di‐cysteine motifs in the enzyme, presenting the entire electron‐transfer pathway and proton‐transfer participants in their native configurations. As such a state cannot generally be enriched and stabilized for analysis, RnQSOX1 gives unprecedented insight into the functional group environments of the four cysteines involved in dithiol/disulfide exchange and provides the framework for analysis of the energetics of electron transfer in the presence of the bound flavin adenine dinucleotide cofactor. Hybrid quantum mechanics/molecular mechanics (QM/MM) free energy simulations based on the X‐ray crystal structure suggest that formation of the interdomain disulfide intermediate is highly favorable and secures the flexible enzyme in a state from which further electron transfer via the flavin can occur.

Path‐integral calculations of heavy atom kinetic isotope effects in condensed phase reactions using higher‐order trotter factorizations

A convenient approach to compute kinetic isotope effects (KIEs) in condensed phase chemical reactions is via path integrals (PIs). Usually, the primitive approximation is used in PI simulations, although such quantum simulations are computationally demanding. The efficiency of PI simulations may be greatly improved, if higher‐order Trotter factorizations of the density matrix operator are used. In this study, we use a higher‐order PI method, in conjunction with mass‐perturbation, to compute heavy‐atom KIE in the decarboxylation of orotic acid in explicit sulfolane solvent. The results are in good agreement with experiment and show that the mass‐perturbation higher‐order Trotter factorization provides a practical approach for computing condensed phase heavy‐atom KIE.

The Role of the M20 Loop in the Hydride Transfer in E. Coli Dihydrofolate Reductase

A key question concerning the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) is whether the Met20 loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the Met20 loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the Met20 loop during the chemical step, we studied the hydride transfer in wild type (WT) ecDHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the Met20 loop in a closed and disordered conformation. Additionally, we investigated three mutant forms (I14X; X = Val, Ala, Gly) of the enzyme that have increased active site flexibility and donor-acceptor distance dynamics in closed and disordered Met20 loop states. We found that the conformation of the Met20 loop has a dramatic effect on the ordering of active site hydration, although the Met20 loop conformation only has a moderate effect on the hydride transfer rate and donor-acceptor distance dynamics. Finally, we evaluated the pKa of the substrate N5 position in closed and disordered Met20 loop states and found a strong correlation between N5 basicity and the conformation of the Met20 loop.