METABOLIC SPECIFICITY OF METHYLTHIOCOFORMYCIN FOR MALARIAL ADENOSINE DEAMINASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 恶性疟原虫是一种嘌呤营养缺陷型生物，需要次黄嘌呤作为关键的代谢前体。红细胞腺嘌呤核苷酸是嘌呤前体的来源，使腺苷脱氨酶(ADA)成为恶性疟原虫次黄嘌呤形成途径中的关键酶。甲硫腺苷(MTA)是大多数疟疾ADA的嘌呤核苷酸底物之一，但不是人类ADA的底物。疟疾ADA的催化位点专一性允许甲硫代考福霉素(MT-Coformycin)作为对人ADA具有低亲和力的疟原虫特异性过渡态类似物[Tyler，P.C.，Taylor，E.A.，Frohlich，R.G.G.和Schramm，V.L.(2007)J.Am化学。SoC。129、6872-6879]。在疟疾ADAs中MTA和MT-Coformycin特异性的结构基础是推测的主题。在这项研究中，我们报道了间日疟原虫ADA(PvADA)与MT-辅酶A形成的络合物的晶体结构，揭示了疟疾ADA中5‘-甲硫基的前所未有的结合几何构型。与疟疾ADA与腺苷或脱氧辅酶A形成的络合物相比，5‘-甲基硫代核糖基团旋转了130度。Asp172和MT-Coformycin的3‘-羟基之间的氢键网络对于识别5’-甲硫代糖基是必不可少的。当MT-Coformycin结合时，水占据了5‘-羟基结合位置。Asp172的突变破坏了MTA和MT-Coformycin的底物专一性。PvADA的动力学、诱变和结构分析，以及其他五种疟原虫ADA的动力学分析，为其对MTA和MT-Coformycin的特异性奠定了独特的结构基础。鸡疟原虫ADA不使用MTA作为底物，不被MT-Coformycin抑制，并且缺失Asp172。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation in Plasmodium falciparum. Methylthioadenosine (MTA) is one of purine nucleotide substrates for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation. In this study, we report the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin that reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172.