Effect of Asp122 Mutation on the Hydride Transfer in E. coli DHFR Demonstrates the Goldilocks of Enzyme Flexibility.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) in the presence of NADPH. The key hydride transfer step in the reaction is facilitated by a combination of enzyme active site preorganization and correlated protein motions in the Michaelis-Menten (E:NADPH:DHF) complex. The present theoretical study employs mutagenesis to examine the relation between structural and functional properties of the enzyme. We mutate Asp122 in Escherichia coli DHFR, which is a conserved amino acid in the DHFR family. The consequent effect of the mutation on enzyme catalysis is examined from an energetic, structural and short-time dynamic perspective. Our investigations suggest that the structural and short-time dynamic perturbations caused by Asp122X mutations (X = Asn, Ser, Ala) are along the reaction coordinate and lower the rate of hydride transfer. Importantly, analysis of the correlated and principle component motions in the enzyme suggest that the mutation alters the coupled motions that are present in the wild-type enzyme. In the case of D122N and D122S, the mutations inhibit coupled motion, whereas in the case of D122A, the mutation enhances coupled motion, although all mutations result in similar rate reduction. These results emphasize a Goldilocks principle of enzyme flexibility, that is, enzymes should neither be too rigid nor too flexible.

Improved Sugar Puckering Profiles for Nicotinamide Ribonucleoside for Hybrid QM/MM Simulations.

The coenzyme nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) play ubiquitous roles as oxidizing and reducing agents in nature. The binding, and possibly the chemical redox step, of NAD+/NADH may be influenced by the cofactor conformational distribution and, in particular, by the ribose puckering of its nicotinamide-ribonucleoside (NR) moiety. In many hybrid quantum mechanics-molecular mechanics (QM/MM) studies of NAD+/NADH dependent enzymes, the QM region is treated by semiempirical (SE) methods. Recent work suggests that SE methods do not adequately describe the ring puckering in sugar molecules. In the present work we adopt an efficient and practical strategy to correct for this deficiency for NAD+/NADH. We have implemented a cost-effective correction to a SE Hamiltonian by adding a correction potential, which is defined as the difference between an accurate benchmark density functional theory (DFT) potential energy surface (PES) and the SE PES. In practice, this is implemented via a B-spline interpolation scheme for the grid-based potential energy difference surface. We find that the puckering population distributions obtained from free energy QM(SE)/MM simulations are in good agreement with DFT and in fair accord with experimental results. The corrected PES should facilitate a more accurate description of the ribose puckering in the NAD+/NADH cofactor in simulations of biological systems.