Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.

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Acyclovir (ACV) has shown efficacy in the prophylactic suppression of human cytomegalovirus (HCMV) reactivation in immunocompromised renal transplant patients without the toxicity associated with ganciclovir (GCV). The HCMV UL97 gene product, a protein kinase, is responsible for the phosphorylation of GCV in HCMV-infected cells. This report provides evidence for the phosphorylation of ACV by UL97. Anabolism studies with the HCMV wild-type strain AD169 and with recombinant mutants derived from marker transfer experiments performed by using mutant UL97 DNA from both clinical isolates and a laboratory-derived strain resistant to GCV showed that mutations in the UL97 gene cripple the ability of recombinant virus-infected cells to anabolize both GCV and ACV. These mutant UL97 recombinant viruses were less susceptible to both GCV and ACV than was the wild-type strain. A recombinant herpes simplex virus type 1 strain, in which the thymidine kinase gene is deleted and the UL13 gene is replaced with the HCMV UL97 gene, was able to induce the phosphorylation of ACV in infected cells. Finally, purified UL97 phosphorylated both GCV and ACV to their monophosphates. Our results indicate that UL97 promotes the selective activity of ACV against HCMV.

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