Wang J, Leblanc E, Chang C F, Papadopoulou B, Bray T, Whiteley J M, Lin S X, Ouellette M
Centre de Recherche en Infectiologie, Québec, Canada.
Arch Biochem Biophys. 1997 Jun 15;342(2):197-202. doi: 10.1006/abbi.1997.0126.
Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in the protozoan parasite Leishmania. Genetic analysis has previously implicated PTR1 in pterin and folate metabolism. PTR1 was purified from a fusion protein expressed in Escherichia coli. Purified PTR1 exhibits NADPH-dependent biopterin, dihydrobiopterin, folate, and dihydrofolate reductase activities. The highest activity was found with the most oxidized pterins. The active protein was found to be a tetramer as demonstrated by gel-filtration chromatography. Kinetic constants (K(m)), as determined by double-reciprocal plots, were calculated for NADPH and for several of PTR1's substrates. The PTR1 of Leishmania tarentolae had a K(m) of 16.9 microM for the cofactor NADPH and K(m) values ranging from 3.5 to 85 microM for the various substrates. The dissociation constant (KD), as determined by fluorescence titration, for NADPH was estimated to be 130 microM. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism of Leishmania should be useful for structure-function analysis and for developing specific inhibitors against this putative important chemotherapeutic target.
短链脱氢酶/还原酶PTR1的过量产生赋予原生动物寄生虫利什曼原虫对二氢叶酸还原酶抑制剂甲氨蝶呤的抗性。遗传分析先前已表明PTR1参与蝶呤和叶酸代谢。PTR1是从在大肠杆菌中表达的融合蛋白中纯化出来的。纯化的PTR1表现出依赖NADPH的生物蝶呤、二氢生物蝶呤、叶酸和二氢叶酸还原酶活性。在最氧化的蝶呤中发现活性最高。通过凝胶过滤色谱法证明活性蛋白是四聚体。通过双倒数作图法测定了NADPH和PTR1的几种底物的动力学常数（K(m)）。大利什曼原虫的PTR1对辅因子NADPH的K(m)为16.9 microM，对各种底物的K(m)值范围为3.5至85 microM。通过荧光滴定法测定的NADPH的解离常数（KD）估计为130 microM。这种参与利什曼原虫叶酸和蝶呤代谢的重要新型酶的生化特性，对于结构-功能分析以及开发针对这个假定的重要化疗靶点的特异性抑制剂应该是有用的。
