X-RAY CRYSTALLOGRAPHIC STUDIES OF CANDIDA-ALBICANS DIHYDROFOLATE-REDUCTASE - HIGH-RESOLUTION STRUCTURES OF THE HOLOENZYME AND AN INHIBITED TERNARY COMPLEX

The recent rise in systemic fungal infections has created a need for t he development of new antifungal agents, As part of an effort to provi de therapeutically effective inhibitors of fungal dihydrofolate reduct ase (DHFR), we have cloned, expressed, purified, crystallized, and det ermined the three-dimensional structure of Candida albicans DHFR. The 192-residue enzyme, which was expressed in Escherichia coli and purifi ed by methotrexate affinity and cation exchange chromatog raphy, was 2 7% identical to human DHFR, Crystals of C. albicans DHFR were grown as the holoenzyme complex and as a ternary complex containing a pyrroloq uinazoline inhibitor. Both complexes crystallized with two molecules i n the asymmetric unit in space group P2(1). The final structures had R -factors of 0.199 at 1.85 Angstrom resolution and 0.155 at 1.60-Angstr om resolution, respectively. The enzyme fold was similar to that of ba cterial and vertebrate DHFR, and the binding of a nonselective diamino pyrroloquinazoline inhibitor and the interactions of NADPH with protei n were typical of ligand binding to other DHFRs. However, the width of the active site cleft of C. albicans DHFR was significantly larger th an that of the human enzyme, providing a basis for the design of poten tially selective inhibitors.