Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii.

Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP+ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of Lactobacillus leichmannii. It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.w value of 0.12 S. The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH2- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group. pCMB inhibited the enzyme activity (IC50 = 2 microM). The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C. It is activated by 0.1 M KCl and KI and 2 M urea. 3-4 M urea completely inactivated the enzyme. Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive.