Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Abstract

Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR•DHF•NADP + catalytic complex, resolved to1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67′-Y69′ residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationallychallenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation. KeywordsR67 DHFR; Dihydrofolate Reductase; X-ray crystallography; ternary complex; Type II DHFR Antifolate drug therapy plays a critical role in the treatment of pathogenic and neoplastic diseases. The evolution of a plasmid-encoded, Type II dihydrofolate reductase (DHFR) provides one mechanism for bacterial evasion of drugs such as trimethoprim that target the bacterial dihydrofolate reductase enzyme (1-4). Type II DHFR is an extremely unusual enzyme that exhibits no apparent structural or evolutionary relationship with the type I (chromosomal) enzyme. It is one of the smallest enzymes known to self-assemble into an active quaternary structure, forming a homotetramer consisting of four 78-residue peptides * To whom correspondence should be addressed. This type of active site structure also creates substantial evolutionary and mutational challenges to the enzyme. Since each mutation will alter four active site residues at a time, most of the evolutionary pressure that would normally optimize enzyme function is compromised by the need to balance the effects of substitutions at all four symmetry-related sites. For example, a residue substitution on one monomer, which might promote folate N5 protonation, may also interfere with NADPH binding when it is prese...

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

“…Arrows on opposite sides of the pore indicate entry of the ligands. In this ternary complex structure (8), NADP ϩ enters from the left and DHF from the right (8); however, binding at symmetry-related sites can also occur. The color code for the atoms is carbon, green; nitrogen, blue; oxygen, red; phosphorus, magenta; and hydrogen, white.…”

“…From NMR, crystallography, and docking studies, the pteridine ring of DHF/folate binds at the center of the pore, but the paraaminobenzoylglutamate tail of dihydrofolate/folate is disordered (5,8,9). Interactions have been predicted between symmetry-related Lys-32 residues (at either edge of the pore) and the glutamate tail of substrate.…”

“…Previous crystallography and NMR studies (8,9,11,12) show NADPH binds in an extended conformation, with numerous specific interactions, including H-bonds between the nicotinamide carboxamide of NADP ϩ with backbone NH and O atoms from Ile-68. Ionic interactions also form between the nicotinamide phosphate and the adenosyl-2Ј-phosphate with symmetry-related Lys-32 residues (13)(14)(15)(16).…”

Structure of the hypothetical DUF1811-family protein GK0453 fromGeobacillus kaustophilusHTA426

“…of 2.2 Å for 52 C atoms; le Maire et al, 2006) and type II R-plasmidencoded R67 dihydrofolate reductase (R67 DHFR; PDB entry 2rh2, chain A; 12% identity; Z-score of 6.5 and r.m.s.d. of 2.7 Å for 51 C atoms; Krahn et al, 2007) (Fig. 2d).…”

“…THF is essential for the synthesis of thymidylate, purine nucleosides, methionine and other metabolic intermediates (Krahn et al, 2007). Since the functional tetramerization and the critical residues for enzymatic function are absent in GK0453, it is unlikely that GK0453 possesses an activity similar to that of DHFR.…”