Division of Biological Chemistry & Drug Discovery, College of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dundee DD1 5EH, Scotland, United Kingdom.
J Biol Chem. 2011 Mar 25;286(12):10429-38. doi: 10.1074/jbc.M110.209593. Epub 2011 Jan 14.
Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H(4)B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity associated with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiological pH and ionic strength and compared with LmQDPR. Specificity constants (k(cat)/K(m)) for LmPTR1 are similar with dihydrobiopterin (H(2)B) and quinonoid dihydrobiopterin (qH(2)B) as substrates and about 20-fold lower than LmQDPR with qH(2)B. In contrast, TbPTR1 shows a 10-fold higher k(cat)/K(m) for H(2)B over qH(2)B. Analysis of Trypanosoma brucei isolated from infected rats revealed that H(4)B (430 nM, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H(4)B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H(4)B as a medium supplement. These cells grew normally with H(4)B, which spontaneously oxidizes to qH(2)B, but were unable to survive in the absence of pterin or with either biopterin or H(2)B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metabolism in African trypanosomes and underline its potential as a drug target.
利什曼原虫寄生虫是蝶呤辅助因子缺陷型生物，它们使用 NADPH 依赖性蝶呤还原酶 1（PTR1）和 NADH 依赖性醌型二氢蝶啶还原酶（QDPR）来回收和维持四氢生物蝶呤（H（4）B）的细胞内池。然而，尽管非洲锥虫的基因组中缺乏可靠的候选 QDPR，但它仍保持着明显的 QDPR 活性。在这里，我们提供的证据表明，其他人以前报道的 NADH 依赖性活性是一种测定假象。我们使用基于 HPLC 的酶测定法，证明与 TbPTR1 和 LmPTR1 相关存在 NADPH 依赖性 QDPR 活性。报告了生理 pH 和离子强度下重组 PTR1 的动力学特性，并与 LmQDPR 进行了比较。LmPTR1 与二氢生物蝶呤（H（2）B）和醌型二氢生物蝶呤（qH（2）B）作为底物的特异性常数（k（cat）/ K（m））相似，而与 qH（2）B 相比，LmQDPR 的特异性常数约低 20 倍。相比之下，TbPTR1 对 H（2）B 的 k（cat）/ K（m）比 qH（2）B 高 10 倍。从感染大鼠中分离出的布氏锥虫的分析表明，H（4）B（430 nM，总生物蝶呤的 98％）是主要的细胞内蝶呤，这与 H（4）B 的回收和再生的双重作用一致。基因敲除实验证实了这一点：只有在 H（4）B 作为培养基补充物过量表达 LmQDPR 的品系中才能获得 PTR1 基因缺失。这些细胞在 H（4）B 存在下正常生长，H（4）B 会自发氧化为 qH（2）B，但在没有蝶呤或培养基中存在生物蝶呤或 H（2）B 的情况下无法存活。这些发现确立了 PTR1 在非洲锥虫的蝶呤代谢中具有重要的双重作用，并强调了它作为药物靶标的潜力。
