Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography

Abstract

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP + . The neutron data were collected to 2.0-Å resolution using a 3.6-mm 3 crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pK a value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.enzyme catalysis | protonation state | protein dynamics | neutron diffraction | deuterium exchange

Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100

“…Mutations of D27, Y100, or both disrupt the sigmoidal pH/rate dependence observed in the WT. The protonation of DHF is likely to occur through a water molecule, as shown by the accompanying crystallographic data by Wan et al (32), whereas the D27 and Y100 residues may help to orient the substrates and the water molecule through various electrostatic interactions. Further evidence of the important role of Y100 was obtained from FEP calculations of the pK a of DHF N5.…”

“…D27 has been shown in previous studies to be important for facilitating a solvent-assisted proton delivery to the N5 position of DHF, thus generating the protonated substrate for the subsequent hydride transfer (4,(16)(17)(18)31). The direct source of the proton is likely to be a water molecule that could enter the active site as the Met20 loop changes conformations (18), and crystal data supporting this hypothesis are provided in the accompanying work by Wan et al (32). Y100 has been implicated to provide electrostatic stabilization of the developing positive charge on the nicotinamide moiety, as well as exhibiting strong electrostatic interactions with NADP + and folate (20,21).…”

Structural Insights into Mycobacterium tuberculosis Rv2671 Protein as a Dihydrofolate Reductase Functional Analogue Contributing to para-Aminosalicylic Acid Resistance

“…In all DHF–DHFR complex structures, a conserved arginine forms an electrostatic interaction with the α -carboxyl group of the glutamate tail. In the E. coli folate-DHFR structure (4pdj), 39 Arg57 corresponds to the conserved arginine in DHFRs. Superimposition of Rv2671 and E. coli folate-DHFR structures put E-coli folate-DHFR Arg57 at the cleft of loop 6 (L6) and loop 7 (L7) in Rv2671 (Figure 2D).…”

Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

“…The atomic arrangements solved by direct methods present a unique opportunity for further improvement of our current estimates of electron scattering factors. Our atomic structures include hydrogen atoms, which play a central role in many biochemical processes; knowing their positions has provided important insights into catalytic mechanisms (33,34) and greatly aids rational drug design (35). Lastly, by using direct methods, MicroED can also provide ab initio solutions for smallmolecule organic compounds, such as rare pharmaceutical polymorphs (36) and designer peptoids (37) that challenge other means of investigation.…”