Tobacco budworm dihydrofolate reductase is a promising target for insecticide discovery

Structural differences in dihydrofolate reductases from different species h ave been exploited to develop specific inhibitory molecules, such as chemot herapeutic agents, antibiotics or antihelminthics, that show species specif icity or selectivity. As dihydrofolate reductase (DHER) is a crucial enzyme for the synthesis of purines, pyrimidines and some amino acids, and also b ecause developing insects show a remarkably rapid rate of cell division, DH FR is a potentially promising target for the discovery of novel insecticide s. We have thus isolated and characterized the enzyme from a serious agricu ltural pest, Heliothis (Helicoverpa) virescens, the tobacco budworm. Sequen cing tryptic peptides of the 35 000-fold purified DHFR allowed the subseque nt isolation of a partial cDNA, with the full Dhfr gene sequence obtained f rom a genomic library. The H. virescens Dhfr spans 4 kb, with three introns , and encodes 185 amino acids. The enzyme shows an overall similarity of ap proximate to 68% with DHER from other metazoans, which has facilitated the molecular modeling of the protein. DHFRs front insects appear to have strik ingly reduced sensitivity to inhibition by methotrexate, compared with the vertebrate enzymes, and this reduction was also reflected in the total bind ing energy seen after modeling experiments. Four residues that may be chara cteristic of insect DHFR, as well as a unique cysteine in the H. virenscens DHER active site, offer insight into the nature of inhibitor selectivity a nd provide suitable target sites for insecticide discovery.