Hydride transfer catalysed by Escherichia coli and Bacillus subtilis dihydrofolate reductase: coupled motions and distal mutations

Abstract

This paper reviews the results from hybrid quantum/classical molecular dynamics simulations of the hydride transfer reaction catalysed by wild-type (WT) and mutant Escherichia coli and WT Bacillus subtilis dihydrofolate reductase (DHFR). Nuclear quantum effects such as zero point energy and hydrogen tunnelling are significant in these reactions and substantially decrease the free energy barrier. The donor-acceptor distance decreases to ca 2.7 A at transition-state configurations to enable the hydride transfer. A network of coupled motions representing conformational changes along the collective reaction coordinate facilitates the hydride transfer reaction by decreasing the donor-acceptor distance and providing a favourable geometric and electrostatic environment. Recent single-molecule experiments confirm that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time-scale of the hydride transfer. Distal mutations can lead to non-local structural changes and significantly impact the probability of sampling configurations conducive to the hydride transfer, thereby altering the free-energy barrier and the rate of hydride transfer. E. coli and B. subtilis DHFR enzymes, which have similar tertiary structures and hydride transfer rates with 44% sequence identity, exhibit both similarities and differences in the equilibrium motions and conformational changes correlated to hydride transfer, suggesting a balance of conservation and flexibility across species.

Figure 1

Three-dimensional structure of E. coli DHFR. The residues conserved across numerous species from E. coli to human are indicated by a gradient colour scheme (grey to red, where red is the most conserved). NADPH and DHF are in green and magenta, respectively. Reproduced with permission from Agarwal et al. (2002b).

Figure 2

The hydride transfer reaction from the NADPH cofactor to the H₃F⁺ substrate.

Figure 3

Selected thermally averaged geometrical properties along the collective reaction coordinate for WT E. coli (left), G121V mutant E. coli (middle) and WT B. subtilis (right) DHFR. From top to bottom, the properties plotted are: the donor–acceptor distance; the angle between the acceptor and methylene amino linkage in H₂F; the distance between C_ζ of Phe31 and C11 of H₂F; the hydrogen-bonding distance between N of Asp122 and O of Gly15 (black) and between O of Ile14 and carboxamide N of NADPH (grey). The relevant residues are labelled in figure 1. The WT E. coli data were obtained from Agarwal et al. (2002b), the G121V mutant E. coli data were obtained from Watney et al. (2003), and the B. subtilis data were obtained from Watney & Hammes-Schiffer (2005). An improved numerical method for calculating the thermally averaged properties was used for the analysis of the B. subtilis data. The B. subtilis results obtained with the numerical method used to generate the E. coli results are provided in Watney (2005), and the qualitative trends are the same.

Figure 4

Free-energy profile of the DHFR-catalysed hydride transfer reaction as a function of a collective reaction coordinate that includes motions of the enzyme, substrate and cofactor. The magnitude of the free-energy barrier is determined by the relative probabilities of sampling the transition state and the reactant configurations. The thermally averaged equilibrium structures, as well as the average donor–acceptor distances in angstroms, are provided for selected values of the reaction coordinate. Note that the donor–acceptor distance decreases as the reaction evolves from the reactant to the transition state. The conformational changes along the collective reaction coordinate are attained by equilibrium thermal motions occurring within the confines of the protein fold and reflect a network of coupled motions extending throughout the protein. This network facilitates the hydride transfer reaction by bringing the donor and acceptor closer together, orienting the substrate and cofactor properly and providing a favourable electrostatic environment.

Figure 5

Electrostatic potential mapped to the molecular surface of the (a) and (b) βF-βG loop and (c) βG-βH loop. The potentials are given for the reactant state (left), transition state (middle) and product state (right). Blue indicates positive potential and red indicates negative potential. The substrate and cofactor are yellow and green, respectively. This figure was created using GRASP (Nicholls et al. 1991). Reproduced with permission from Wong et al. (2004).

Figure 6

Three-dimensional structure of WT E. coli DHFR with the NADPH cofactor shown in green and the substrate shown in magenta. The important loop regions are identified and the residues involved in the mutant DHFR enzymes studied are labelled with red spheres. Reproduced with permission from Hammes-Schiffer (2006).

Figure 7

Thermally averaged structures for E. coli (red) and B. subtilis (blue) DHFR at the reactant state (RS), transition state (TS) and product state (PS). Reproduced with permission from Watney & Hammes-Schiffer (2005).