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