Identification of endogenous ligands bound to bacterially expressed human and E. coli dihydrofolate reductase by 2D NMR.

Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed "humanized" mutant of ecDHFR switches binding specificity in the cell.