The Evolution of Function within the Nudix Superfamily: Input file

The Nudix superfamily encompasses over 80,000 protein domains from all three domains of life. These proteins fall into four general functional classes: isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch-specific adenine glycosylases (A/G-specific adenine glycosylases), pyrophosphohydrolases, and non-enzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally-based annotations. We manually constructed structure-guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 “select” Nudix domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix clan revealed general monophyly with regard to function and a few instances of probable homoplasy.

Nudix超家族（Nudix superfamily）涵盖了生命三大域的8万余个蛋白质结构域。该超家族的蛋白质可划分为四大功能类别：异戊烯基二磷酸异构酶（isopentenyl diphosphate isomerases，IDIs）、腺嘌呤/鸟嘌呤错配特异性腺嘌呤糖苷酶（A/G特异性腺嘌呤糖苷酶）、焦磷酸水解酶，以及诸如蛋白质-蛋白质相互作用、转录调控在内的非酶活性。其中规模最大的类群为焦磷酸水解酶，涵盖超过100种不同的水解酶底物特异性。为阐明该家族如此丰富的功能活性的演化机制，我们从已发表文献中收集了205个Nudix蛋白质的实验与结构数据，并进行组装与分析。我们修正了基因本体注释（Gene Ontology Annotation）数据库中该家族53条存在功能错误的注释，或为其补充了更为恰当的描述，并提出了275条基于实验证据的全新注释。我们手动构建了78个Nudix蛋白质的结构引导序列比对。以该结构比对作为参考种子，我们进一步对347个“精选”Nudix结构域开展了序列比对；这些结构域均经过结构解析、功能鉴定或系统发育层面具有重要意义的筛选。基于对Nudix焦磷酸水解酶结构与底物特异性的综述分析，我们进一步研究了Nudix水解酶基序下游的一个环区；该区域此前被证实可与底物分子结合，且携带已知的功能基序。该环区为水解酶家族内的功能辐射与底物特异性演化提供了潜在的结构基础。最后，我们对347个精选蛋白质结构域以及完整的Nudix家族簇开展了系统发育分析，结果显示该家族的功能大多符合单系群特征，仅存在少数疑似同塑性演化的案例。