The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system in studies of the role of protein dynamics in catalysis, but so far these studies were limited by the availability of structural information. Second, CbFDH and its mutants are of use in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in its commercial development. Here, we report the crystallization and structural determination of both holo-CbFDH and apo-CbFDH. The free energy barrier for the catalyzed reaction is 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 apo-enzyme crystal was obtained under co-crystallization conditions with a saturating concentration of both the cofactor (NAD+) and inhibitor (azide), which has a nM dissociation constant. It was found that the fraction of the apo-enzyme present in the solution is less than 1.7x10−7 (i.e. the solution is 99.9999% holo-enzyme). This is an extreme case where the crystal structure represents an insignificant fraction of enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

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 towards the design of enzymes whose catalytic power is closer to that of native enzymes than to that of the current generation of designer enzymes.

A key question concerning the catalytic cycle of dihydrofolate reductase (DHFR) is whether the Met loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the Met loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the Met loop during the chemical step, we studied the hydride transfer in wild type (WT) DHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the Met loop in a closed and disordered conformation. Additionally, we investigated three mutant forms (I14; = Val, Ala, Gly) of the enzyme that have increased active site flexibility and donor-acceptor distance dynamics in closed and disordered Met loop states. We found that the conformation of the Met loop has a dramatic effect on the ordering of active site hydration, although the Met loop conformation only has a moderate effect on the hydride transfer rate and donor-acceptor distance dynamics. Finally, we evaluated the p of the substrate N5 position in closed and disordered Met loop states and found a strong correlation between N5 basicity and the conformation of the Met loop.

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.