Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia.
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia.
Nat Commun. 2018 Jun 20;9(1):2411. doi: 10.1038/s41467-018-04669-9.
Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of diverse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the "marker of ligase activity", together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.
天冬酰胺内肽酶（AEPs）是一类在植物防御和种子贮藏蛋白成熟过程中起关键作用的蛋白酶。然而，一些特定的植物 AEP 并非作为蛋白酶发挥作用，而是作为转肽酶（连接酶），催化肽末端的分子内连接，从而导致肽环化。这些连接酶型 AEP 在体外肽工程到植物分子农业等领域具有潜在的生物技术应用，但目前尚不清楚使这些酶能够催化肽连接/环化而不是蛋白水解的结构特征。在这里，我们通过结合分子建模、序列空间分析和在植物体内的功能测试，比较了不同植物 AEP 的序列、结构和功能。我们发现，底物结合口袋内以及相邻环中的变化，在这里称为“连接酶活性标志物”，共同对 AEP 连接酶效率起着关键作用。鉴定这些结构决定因素可能有助于发现更多的连接酶型 AEP，并对具有定制催化特性的 AEP 进行工程改造。
