Table1_Structural and evolutionary insights into astacin metallopeptidases.xlsx

The astacins are a family of metallopeptidases (MPs) that has been extensively described from animals. They are multidomain extracellular proteins, which have a conserved core architecture encompassing a signal peptide for secretion, a prodomain or prosegment and a zinc-dependent catalytic domain (CD). This constellation is found in the archetypal name-giving digestive enzyme astacin from the European crayfish Astacus astacus. Astacin catalytic domains span ∼200 residues and consist of two subdomains that flank an extended active-site cleft. They share several structural elements including a long zinc-binding consensus sequence (HEXXHXXGXXH) immediately followed by an EXXRXDRD motif, which features a family-specific glutamate. In addition, a downstream SIMHY-motif encompasses a “Met-turn” methionine and a zinc-binding tyrosine. The overall architecture and some structural features of astacin catalytic domains match those of other more distantly related MPs, which together constitute the metzincin clan of metallopeptidases. We further analysed the structures of PRO-, MAM, TRAF, CUB and EGF-like domains, and described their essential molecular determinants. In addition, we investigated the distribution of astacins across kingdoms and their phylogenetic origin. Through extensive sequence searches we found astacin CDs in > 25,000 sequences down the tree of life from humans beyond Metazoa, including Choanoflagellata, Filasterea and Ichtyosporea. We also found < 400 sequences scattered across non-holozoan eukaryotes including some fungi and one virus, as well as in selected taxa of archaea and bacteria that are pathogens or colonizers of animal hosts, but not in plants. Overall, we propose that astacins originate in the root of Holozoa consistent with Darwinian descent and that the latter genes might be the result of horizontal gene transfer from holozoan donors.

星形粘菌素（astacins）是一类金属肽酶（metallopeptidases, MPs）家族，目前已在多种动物中得到广泛研究与系统阐释。该家族成员均为多结构域胞外蛋白，其保守核心架构包含分泌信号肽、前结构域（或称前肽段）以及锌依赖型催化结构域（catalytic domain, CD）。这一结构组合存在于经典的命名来源消化酶——欧洲螯虾（Astacus astacus）源星形粘菌素中。

星形粘菌素的催化结构域全长约200个氨基酸残基，由两个亚结构域组成，二者环绕形成延伸的活性位点裂隙。该家族催化结构域共享多项结构特征：包括一段长锌结合保守序列（HEXXHXXGXXH），其紧邻下游存在EXXRXDRD基序，该基序包含一个家族特异性的谷氨酸残基；此外，其下游的SIMHY基序还包含一个“Met-turn”甲硫氨酸残基与一个锌结合酪氨酸残基。

星形粘菌素催化结构域的整体架构与部分结构特征，与其他亲缘关系较远的金属肽酶高度一致，二者共同构成了金属肽酶的metzincin族。本研究进一步分析了前结构域（PRO-）、MAM结构域、TRAF结构域、CUB结构域以及EGF样结构域的三维结构，并阐明了它们关键的分子决定簇。

此外，本研究还探究了星形粘菌素在各生物界的分布情况及其系统发育起源。通过大规模序列检索，本研究在生命树中超过25000条序列里发现了星形粘菌素催化结构域，涵盖从人类所在的后生动物（Metazoa）到后生动物以外的类群，包括领鞭毛虫门（Choanoflagellata）、丝足虫类（Filasterea）以及鱼孢霉纲（Ichtyosporea）。本研究同时在不足400条序列中发现了该家族成员，它们散见于非全动物真核生物类群（包括部分真菌与一种病毒），以及部分可作为动物宿主病原菌或定植菌的古菌与细菌类群中，但未在植物中检测到星形粘菌素的存在。

综上，本研究提出星形粘菌素起源于全动物界（Holozoa）的根部，符合达尔文式演化路径；而前述非全动物类群中的星形粘菌素基因，可能来源于全动物供体的水平基因转移事件。