The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity
The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity
复制标题
DOI：
10.1101/380295
复制
发表时间：
2018-07
期刊：
影响因子：
--
通讯作者：
A. James;J. Haywood;J. Leroux;K. Ignasiak;Alysha G. Elliott;J. Schmidberger;Mark F. Fisher;S. Nonis;R. Fenske;C. Bond;J. Mylne
中科院分区：
文献类型：
--
作者：
A. James;J. Haywood;J. Leroux;K. Ignasiak;Alysha G. Elliott;J. Schmidberger;Mark F. Fisher;S. Nonis;R. Fenske;C. Bond;J. Mylne
植物天冬酰胺内肽酶（AEPs）是一种无活性的酶原，在贮藏液泡的低pH环境中，通过裂解依赖的自激活来完成种子贮藏蛋白的成熟。AEPs因其大环化反应而备受关注，并被归类为裂解或结扎专家。然而，我们最近发现AEPs产生环或非环产物的能力可以通过活性位点区域的突变而改变，并且在良好的pH条件下，一些AEPs能够进行大环化。从阴蒂种子中提取的一种AEP（丁酸酶1）被归类为连接酶而不是蛋白酶，这为测试切割活性的丧失提供了机会。通过构建重组丁酶1并挽救缺乏AEP的拟南芥突变体，我们发现丁酶1在体内和体外都保留了裂解功能。体内恢复是不完整的，这与丁酸酶1特化向大环化的一些权衡是一致的。其晶体结构显示出一个与切割AEPs仅存在细微差异的活性位点，提示其肽结合区域的诸多差异是其高效大环化的来源。综上所述，似乎要么是丁酸酶1还没有完全特化，要么是对自催化裂解的要求是对AEPs大环化的进化限制。
Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs have attracted attention for their macrocyclization reactions and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.