STRUCTURE STUDIES OF MIMIVIRUS CAPPING ENZYMES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 拟病毒是一种最近发现的寄生于多食阿米巴的寄生虫，它具有非常大的基因组（1.2 Mb），编码911个预测蛋白。拟病毒蛋白质组包含大量在DNA和RNA代谢中具有估算功能的酶。拟病毒似乎编码其自身的mRNA合成机制，其包括多亚基RNA聚合酶和类似于牛痘病毒D1蛋白的推定RNA加帽酶。这些特征突出了拟病毒基因表达策略将具有痘病毒样风味的可能性。因此，我们研究了拟病毒加帽酶的生化特性，以及它与牛痘加帽机制的相似或不同程度。 我们已经纯化、测定和结晶了三功能拟病毒mRNA加帽酶的残基（1-237），该酶包含最小的自主三磷酸酶结构域。 在四个预测的金属结合谷氨酸（Glu 37，Glu 39，Glu 212，Gl 214）中的每一个处的丙氨酸取代消除了MimiCE的NTP磷酸水解酶活性，从而验证了其在金属依赖性病毒/真菌/原生动物三磷酸酶家族中的成员资格。 因此，mimivirus加帽酶的小的三磷酸酶结构域（我们可以从细菌中以50 mg的量纯化）为病毒金属依赖性RNA三磷酸酶蛋白的研究提供了易于处理的靶。 基于痘病毒加帽酶和拟病毒的加帽酶之间的广泛相似性，在拟病毒加帽三磷酸酶的研究中获得的观察结果应该适用于痘病毒，因此，在结构促进的药物设计中有很大的用途。

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. Mimivirus is a recently identified parasite of Acanthamoeba polyphaga that has an extraordinarily large genome (1.2 Mb) encoding 911 predicted proteins. The mimivirus proteome embraces a slew of enzymes with imputed functions in DNA and RNA metabolism. Mimivirus appears to encode its own mRNA synthetic machinery, which includes a multisubunit RNA polymerase and a putative RNA capping enzyme that resembles the vaccinia virus D1 protein. These features highlight the likelihood that the mimivirus gene expression strategy will have a poxvirus-like flavor. We therefore examined the biochemical properties of the mimivirus capping enzyme and the extent to which it resembles or differs from the vaccinia capping machinery. We have purified, assayed, and crystallized residues (1-237) of the tri-functional mimivirus mRNA capping enzyme which comprises a minimal autonomous triphosphatase domain. Alanine substitutions at each of the four predicted metal-binding glutamates (Glu37, Glu39, Glu212, Gl214) abolished the NTP phosphohydrolase activity of MimiCE, thereby verifying its membership in the metal-dependent viral/fungal/protozoal triphosphatase family. Thus, the small triphosphatase domain of the mimivirus capping enzyme (which we can purify from bacteria in 50 mg quantities) presents a tractable target for the investigation of viral metal-dependent RNA triphosphatase proteins. Based on extensive similarities between poxvirus capping enzymes and that of mimivirus, the observations gained in the study of the mimivirus capping triphosphatase should be applicable to poxvirusus at large and, therefore, of great use in structure-facilitated drug design.