NMR Structures of Apo L. casei Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

bS Supporting Information The enzyme dihydrofolate reductase (DHFR; 5,6,7,8-tetra-hydrofolate:NADPH oxidoreductase, EC 1.5.1.3) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydro-folate (THF) using NADPH as coenzyme.1 Since THF and its metabolites are precursors of purine and pyrimidine bases, the normal functioning of this enzyme is essential for proliferating cells. This makes DHFR an excellent target for antifolate drugs such as methotrexate (anticancer), pyrimethamine (antimalarial), and trimethoprim (antibacterial). Such agents act by inhibiting the enzyme in parasitic or malignant cells.1,2 The cooperative binding of ligands to DHFR plays an important role not only in the enzyme catalytic cycle (negative cooperativity in THF/ NADPH binding)3 but also in enzyme inhibition (positive cooperativity in antifolate/NADPH binding).4 The effects of positive cooperative binding in controlling enzyme inhibition ar

New gene cassettes for trimethoprim resistance, dfr13, and Streptomycin-spectinomycin resistance, aadA4, inserted on a class 1 integron

In a previous survey of 357 trimethoprim-resistant isolates of aerobic gram-negative bacteria from commensal fecal flora, hybridization experiments showed that 25% (90 of 357) of the isolates failed to hybridize to specific oligonucleotide probes for dihydrofolate reductase types 1, 2b, 3, 5, 6, 7, 8, 9, 10, and 12. Subsequent cloning and sequencing of a plasmid-borne trimethoprim resistance gene from one of these isolates revealed a new dihydrofolate reductase gene, dfr13, which occurred as a cassette integrated in a site-specific manner in a class 1 integron. The gene product shared 84% amino acid identity with dfr12 and exhibited a trimethoprim inhibition profile similar to that of dfr12. Gene probing experiments with an oligonucleotide probe specific for this gene showed that 12.3% (44 of 357) of the isolates which did not hybridize to probes for other dihydrofolate reductases hybridized to this probe. Immediately downstream of dfr13, a new cassette, an aminoglycoside resistance gene of the class AADA [ANT(3")(9)-I], which encodes streptomycin-spectinomycin resistance, was identified. This gene shares 57% identity with the consensus aadA1 (ant(3")-Ia) and has been called aadA4 (ant(3")-Id). The 3′ end of the aadA4 cassette was truncated by IS26, which was contiguous with a truncated form of Tn3. On the same plasmid, pUK2381, a second copy of IS26 was associated with sul2, which suggests that both integrase and transposase activities have played major roles in the arrangement and dissemination of antibiotic resistance genes dfr13, aadA4, bla(TEM-1), and sul2

Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-Resistant Streptococcus pneumoniae

Trimethoprim resistance in Streptococcus pneumoniae can be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC(50)], 4.2 μM) than was the DHFR from strain CP1015 (IC(50), 0.09 μM). However, K(m) values indicated a lower affinity for the enzyme's natural substrates (K(m) for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, K(m) values for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC(50) of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates

Refolding of recombinant Pneumocystis carinii dihydrofolate reductase and characterization of the enzyme.

The isolation of dihydrofolate reductase (DHFR) cDNA sequences from the messenger RNA of Pneumocystis carinii using the polymerase chain reaction is described. The 206-amino acid P. carinii DHFR was expressed to high levels in Escherichia coli inclusion bodies using the T7 promoter expression system. Solubilization of the inclusion bodies in 4 M guanidine hydrochloride and refolding of the recombinant protein in the presence of 0.5% polyethylene glycol 1450 yielded correctly folded DHFR which was purified to homogeneity by methotrexate-Sepharose affinity chromatography. The refolded enzyme was readily crystallized as a ternary complex with NADPH and various inhibitors. The enzyme exhibited a sharp pH optimum with maximum activity at pH 7.0 (turnover number = 6500 min-1). Km values for dihydrofolate (DHF) and NADPH were 2.3 and 3.0 microM, respectively, in 0.1 m imidazole buffer, pH 7. Folate did not act as a substrate. Comparison of the kinetic properties of the refolded enzyme with soluble P. carinii DHFR expressed at low levels in the T7 expression system showed similar pH-activity profiles, Km values for DHF and NADPH, and IC50 values for several known antifolates which were tested as inhibitors of the enzyme