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首页> 外文期刊>Catalysis science & technology >Sortase A catalyzed reaction pathways: a comparative study with six SrtA variants
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Sortase A catalyzed reaction pathways: a comparative study with six SrtA variants

机译：排序酶A催化反应途径：具有六个SRTA变体的比较研究

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摘要

Sortase A from Staphylococcus aureus (SrtA) has emerged as a useful enzymatic tool to covalently link proteins in a site-directed manner. Despite the proposed specificity of the SrtA-catalyzed reaction for target proteins carrying the amino acid motif LPXTG, enzymatic side reactions are frequently encountered impairing the yield and purity of the transpeptidation product. In this comparative study we investigated reactions of six different variants of SrtA with soluble protein substrates. Besides the transpeptidation reaction generating the desired heterodimeric product, all tested SrtA variants also catalyzed the formation of byproducts [i.e. homooligomeric and hydrolyzed species of the target protein) to different extents. We found that not only the choice of the enzyme, but also the composition and the context of the sorting motif within the particular amino acid sequence of the target protein had a critical impact on the apparent initial rates of the coupling reactions. In addition to the natural sorting signal LPETG, the-fnotif LAETG was also recognized by the investigated SrtA variants. Even though the overall coupling rates with the LAETG-containing target proteins were rather slow, the formation of unwanted byproducts was largely suppressed under these conditions.
机译：来自金黄色葡萄球菌（SRTA）的分类酶A已成为一种有用的酶促工具，可以以站点定向的方式共价连接蛋白质。尽管提出了针对携带氨基酸基序LPXTG的靶蛋白的SRTA催化反应的特异性，但经常遇到酶促副反应，从而损害了转肽产物的产率和纯度。在这项比较研究中，我们研究了SRTA六种不同变体与可溶性蛋白底物的反应。除了产生所需的异二聚体产物的转肽反应外，所有测试的SRTA变体还催化了副产物的形成[即靶蛋白的家族冷凝物和水解物种）到不同的范围。我们发现，不仅选择酶的选择，还可以选择靶蛋白的特定氨基酸序列中的分选基序的组成和上下文对偶联反应的明显初始速率产生了关键影响。除了自然分类信号LPETG外，研究的SRTA变体还认识到了Fnotif LaETG。尽管与含LAETG靶蛋白的总体耦合率相当缓慢，但在这些条件下，不需要的副产物的形成在很大程度上被抑制了。

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