Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1.
Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1.
复制标题
塔伦托利什曼原虫 H 基因座短链脱氢酶/还原酶 PTR1 减少蝶呤和叶酸。
DOI：
10.1006/abbi.1997.0126
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发表时间：
1997
影响因子：
3.9
通讯作者：
Ouellette,M
中科院分区：
文献类型：
--
作者：
Wang,J;Leblanc,E;Chang,CF;Papadopoulou,B;Bray,T;Whiteley,JM;Lin,SX;Ouellette,M
短链脱氢酶/还原酶PTR1的过量产生使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产生抗性。从大肠杆菌表达的融合蛋白中纯化PTR1，纯化的PTR1具有NADPH依赖的生物蝶呤、二氢生物蝶呤、叶酸和二氢叶酸还原酶活性。最高的活性被发现与最氧化的蝶呤。凝胶过滤层析证实活性蛋白为四聚体。动力学常数（Km），由双倒数图确定，计算NADPH和PTR1的几个底物。利什曼原虫PTR1对辅酶NADPH的Km值为16.9 μ m，对各种底物的Km值为3.5~85 μ m。通过荧光滴定法测定的NADPH解离常数（KD）估计为130 μ m。这一重要的和新的酶参与叶酸和蝶呤代谢ofLeishmaniashould的生化特性是有用的结构-功能分析和开发针对这一假定的重要化疗靶点的特异性抑制剂。
Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in the protozoan parasiteLeishmania.Genetic analysis has previously implicated PTR1 in pterin and folate metabolism. PTR1 was purified from a fusion protein expressed inEscherichia 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 (Km), as determined by double-reciprocal plots, were calculated for NADPH and for several of PTR1's substrates. The PTR1 ofLeishmania tarentolaehad aKmof 16.9 μmfor the cofactor NADPH andKmvalues ranging from 3.5 to 85 μmfor the various substrates. The dissociation constant (KD), as determined by fluorescence titration, for NADPH was estimated to be 130 μm. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism ofLeishmaniashould be useful for structure–function analysis and for developing specific inhibitors against this putative important chemotherapeutic target.