Isomorphous Crystal Structures of Escherichia coli Dihydrofolate Reductase Complexed with Folate, 5-Deazafolate, and 5,10-Dideazatetrahydrofolate: Mechanistic Implications

Abstract

Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does not favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR.NADPH complex (space group P3221; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C.(ABSTRACT TRUNCATED AT 400 WORDS)

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

“…1 A and B); these conformational fluctuations are consistent with single-molecule fluorescence results (19,21). Furthermore, MTX binds in an orientation in which the pterin ring is flipped 180°with respect to substrate, resulting in the MTX N1 atom being placed within H-bonding distance of the catalytic D27 residue (20,22).…”

Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands

“…S1A). Also, as seen in X-ray crystal structures of DHFR bound with THF analogs, the severe puckering of the pterin ring in E: THF promotes formation of a hydrogen bond network with ideal geometry between the carbonyl oxygen of the pterin ring, an invariant water molecule, and N ε1 of Trp22 (10,(20)(21)(22). The difference in Trp22 15 N ε1 chemical shift between the E:THF and E: FOL complexes (Δδ ¼ 2.0 ppm) and between the E:THF and E:DHF complexes (Δδ ¼ 1.8 ppm) is consistent with N ε1 of Trp22 forming a stronger hydrogen bond (23) in the product binary complex compared to the substrate binary complexes.…”

“…These motional differences appear to arise from the differing interactions made by the pterin ring with the Met20 loop. The highly puckered pterin ring of THF allows formation of an ideal water-bridged hydrogen bond network (21,22). This stabilizing hydrogen bond network is likely to alter the conformational dynamics of the Met20 and neighboring active site loops, and also appears to play a direct role in decreasing the dissociation rate of THF relative to DHF substrate (20)(21)(22)30).…”

“…1A). In solution, the enzyme accesses two major conformations, closed and occluded, that differ primarily in the structure of the active site Met20 loop (residues [16][17][18][19][20][21][22] and the adjoining FG (residues 119-123) and GH (residues 142-149) loops (10,11). In the closed conformation, the Met20 loop packs tightly against the nicotinamide ring of the cofactor bound within the active site, as seen in the NADPH complex (E:NADPH) and the ternary complex with the substrate analog folate (FOL) and nicotinamide adenine dinucleotide phosphate oxidized form (NADP þ ) (E:FOL: NADP þ ); the latter is considered to be a model for the Michaelis complex (10).…”

Structure and Site-directed Mutagenesis of a Flavoprotein fromEscherichia coli That Reduces Nitrocompounds

“…These two residues were considered to be candidates for NADPH binding in the FRP crystal structure (21 recognizes NADPH considering the possibility of refolding of the flexible loop in solution, although Arg 208 is not directed toward the active site in the present structure. As observed in other NADPH binding enzymes, there are examples where positively charged residues, such as arginine, lysine, and histidine, are involved in the recognition of the 2Ј-phosphate group of NADPH through a hydrogen bond (41)(42)(43)(44)(45). Thus, if Arg 203 or Arg 208 interact with NADPH, mutation of these residues will contribute to a decrease in NADPH binding affinity to NfsA.…”