Dihydrofolate reductase created by Michael Kim
Dihydrofolate reductase (DHFR) from Escherichia coli (PDB ID = 1RX2) is a housekeeping enzyme that catalyzes the reduction of 7, 8-dihydrofolate (DHF) by NADPH to 5, 6, 7, 8-tetrahydrofolate (THF) (1). Researchers are interested in characterizing this enzyme because THF is used in the synthesis of the nucleoside base thymidine, and by extension, cell proliferation. Inhibition of DHFR can slow the rate of cell proliferation, and DHFR is a drug target in cancerous cells, where cell proliferation is rapid and unregulated. The calculated molecular weight of DHFR is 18,000.36 Da, and the isoelectric point is 4.76 (2).
In E. coli, DHFR functions as a monomeric enzyme, and contains an adenosine binding domain and loop domain (3). The secondary structure of DHFR is comprised of an eight stranded β-sheet, with strands labeled βA – βH, four α-helices (B, C, E, F), and four short mobile loops (1). The adenosine binding domain consists of β strands B (residues 39 – 43), C (residues 58 – 63), D (residues 73 – 75), and E (residues 89 – 95), as well as helices C (residues 43 – 50), E (residues 77 – 86), and F (residues 96 – 104) (4). The loop domain contains three loops: the M20 loop (residues 10 – 24), F-G loop (residues 117 – 131), and G-H loop (residues 146 – 148) (5).
The PDB entry for E. coli DHFR includes four ligands: NADP+, folic acid, manganese2+ (Mn(II)), and β-mercaptoethanol. Mn(II) and β-mercaptoethanol were used to produce isomorphous protein crystal structures, and do not play a role in the biological function of DHFR. NADP+ is the oxidized form of NADPH, the cofactor that serves as the hydride donor to the substrate, DHF. Folic acid is the structural precursor to DHF, which has two additional hydrogen atoms, as well as THF, which has four additional hydrogen atoms.The adenosine binding domain binds the cofactor NADPH, and the loop domain contains the substrate, DHF, and these two domains facilitate hydride transfer from C-4 of NADPH to C-6 of DHF and protonation of N-5 of DHF (6).
The reaction pathway catalyzed by DHFR can be split into five distinct kinetic intermediates: a holoenzyme form bound to NADPH, a Michaelis complex with NADPH and DHF, a transition state complex, a THF binary complex, and a THF-NADPH complex (7). The first three kinetic intermediates correspond to a ‘closed’ conformation, in which the Met-20 loop covers the active site of the enzyme, protecting the substrate from solvent interaction. The center of the Met-20 loop (residues 16 – 19) form a hairpin turn, and Asn-18(N-eta-1) hydrogen bonds with His-45(O) of helix C, stabilizing this intermediate structure (1). Ile-14(O) of the Met-20 loop also hydrogen bonds to the nicotinamide carboxamide group in NADPH. Van der Waals interactions between Asn-18, Ala-19, and the R group of Met-20 with the ribose of NADPH further stabilize this conformation. The N terminal of the Met-20 loop also forms two hydrogen bonds with the F-G loop, between Gly-15(O) and Asp-122(N), and Glu-17(N) and Asp-122(O-eta-2).
The ‘occluded’ conformation corresponds to the last two kinetic intermediates of the reaction pathway. In this conformation, the Met-20 loop extends into the nicotinamide binding pocket, disrupting the binding of enzyme to NADPH. Residues 17 – 20 of the Met-20 loop form a 310 helix, and Met-16 engages in Van der Waals interactions with Thr-46 and Ser-49 of helix C. The hydrogen bonds between the N-terminal of the Met-20 loop and the F-G loop are disrupted, and the C-terminal of the Met-20 loop form a pair of hydrogen bonds with the G-H loop, between Asn-23(O) and Ser-148(N), and Asn-23(N) and Ser-148(O-gamma).
In the reaction pathway, DHF fits into the para-aminobenzoyl glutamate (pABG) binding pocket of DHFR, and in the transition state, the pABG region is sandwiched in between Ile-50 of helix C, and Leu-28 of helix B. A larger portion of the pABG region is surrounded by the hydrophobic side chains of Leu-28, Phe-31, Ile-50, and Leu-54 (8). The nicotinamide region of NADPH is brought closer to the pteridine ring of DHF through rotation between the adenosine binding domain and the loop domain, allowing hydride transfer and protonation of N5 of DHF to occur (6). Hydrogen bonding between the pyrophosphate oxygen atoms of NADPH and N-terminal amide nitrogen atoms of helix B stabilizes the close interaction of the nicotinamide and pteridine rings (8).
Methotrexate (MTX) is a competitive inhibitor of DHFR, and binds tightly in the pABG pocket that normally fits DHF (PDB ID = 1RX3). The protein-drug complex is similar to the protein-substrate complex, as MTX and DHF are structurally similar. DHF cannot hydrogen bond to the carbonyl oxygen atoms of Leu-5(O) and Ile-94(O), but MTX can, and this partly contributes to the higher binding affinity of MTX to DHFR (8). As a competitive inhibitor, MTX binds to the active site of DHFR with higher affinity, and prevents the conversion of DHF into THF, which is required for the synthesis of the nucleoside thymidine. Without thymidine, cells cannot synthesize DNA, and are not able to divide and proliferate. Methotrexate is used as an anticancer drug to prevent cancer cells from undergoing dangerous and unregulated proliferation.
DHFR from Bacillus anthracis (PDB ID = 3E0B) is similar to DHFR in E. coli in both primary and secondary strucutre; it has a calculated PSI-BLAST E value of 4e-88, indicating a high degree of amino acid sequence similarity (9). It also has a Dali Z score of 26.6, which corresponds to a high degree of tertiary structural similarity (10). This particular DHFR is sensitive to the nonselective inhibitor MTX, but resistant to trimethoprim (TMP), a selective DHFR inhibitor for E. coli and Staphylococcus aureus (11). Finding a selective DHFR inhibitor for B. anthracis could reduce the threat of anthrax as a biological weapon. DHFR from B. anthracis exhibits 62% sequence matching in the active site region and 39% sequence matching overall when compared with DHFR from E. coli.
The function, structure, and ligands of DHFR in B. anthracis are almost identical to DHFR in E. coli. Both enzymes have a secondary structure comprised of four α-helices and an eight-stranded β-sheet, and both bind NADPH and DHF in similar binding pockets (12). The ligand referenced in the PDB ID of this DHFR is meant to be a competitive inhibitor, similar to MTX, but specific to anthrax, and not humans. Compared to the DHFR from E. coli, the hydrophobic pABG binding pocket of DHFR from B. anthracis contains Asn-47 in place of Thr-46, Ala-50 in place of Ser-49, and conserves Ile-51 and Leu-55. The changes in the binding domain make the B. anthracis DHFR susceptible to folate analog inhibitors that are not specific for DHFR from other species.