Mutant PTR1 Proteins from Leishmania tarentolae: Comparative Kinetic Properties and Active-Site Labeling

PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3). Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE–Trisacryl. The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor. 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (Ki ≈ 38 → 3 nM) against the pterin substrate and both NADP+ and folate are inhibitors although somewhat weaker. Dihydropteridines are poor substrates compared to the fully oxidized pteridine. Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP+ allows the formation of ternary nonproductive complexes with folate. The kinetic results are consistent with a sequential ordered bi–bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme. Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)3K motif. In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity. Two Y(Xaa)3K regions are present in wild-type PTR1 at 152 and 194. Only Y194F gives protein with zero activity. This observation coupled with affinity labeling of PTR1 by oNADP+ (2′,3′-dialdehyde derivative of NADP+) followed by NaBH4 reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194. The mutation K198Q eliminates inactivation by oNADP+ supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family.