Major objectives of this research are to (1) isolate loo mg of homogeneous dihydrofolate reductase (DHFR) from Pseudomonas aeruginosa, (2) crystallize the enzyme, and (3) determine its three-dimensional structure using X-ray diffraction methods. Armed with the resulting detailed knowledge of the geometrical and topographical properties of Pseudomonas DHFR in relation to the known structure of the corresponding vertebrate enzyme, Agouron Technology will design specific, pharmacologically active inhibitors that selectively cripple the bacterial reductase without interfering with one-carbon metabolism in the host. Such novel inhibitors will address the current critical need for new antimicrobics with enhanced activity against Pseudomonas aeruginosa.Intermediate objectives of Phase I research are to:(1) isolate P. aeruginosa DHFR of sufficient purity and in sufficient quantity for biochemical characterization and for protein sequence determination;(2) characterize the interaction of purified P. aeruginosa DHFR with the antifolate trimethoprim (TMP) in terms of absolute affinity and Ki for the DHF THF conversion; and(3) clone the structural gene for P. aeruginosa DHFR.National Institute of Allergy and Infectious Diseases (NIAID)
