Comparative Analysis and Ancestral Sequence Reconstruction of Bacterial Sortase Family Proteins Generates Functional Ancestral Mutants with Different Sequence Specificities
Comparative Analysis and Ancestral Sequence Reconstruction of Bacterial Sortase Family Proteins Generates Functional Ancestral Mutants with Different Sequence Specificities
复制标题
DOI：
10.3390/bacteria1020011
复制
发表时间：
2022-06
期刊：
影响因子：
--
通讯作者：
Jordan D. Valgardson;Sarah A. Struyvenberg;Zachary R. Sailer;I. Piper;Justin E. Svendsen;D. A. Johnson;Brandon A. Vogel;John M. Antos;M. Harms;J. Amacher
中科院分区：
文献类型：
--
作者：
Jordan D. Valgardson;Sarah A. Struyvenberg;Zachary R. Sailer;I. Piper;Justin E. Svendsen;D. A. Johnson;Brandon A. Vogel;John M. Antos;M. Harms;J. Amacher
革兰氏阳性菌是已知最早的生命形式之一，在 20 亿年前就与革兰氏阴性菌不同。这些生物体利用分选酶将蛋白质附着到其肽聚糖细胞壁上，这是区分两种细菌的结构特征。分选酶的转肽酶活性使其成为蛋白质工程应用中的重要工具，例如在分选酶介导的连接或分选中。然而，由于催化效率相对较低，人们正在不断努力为这些用途创造更好的分选酶变体。在这里，我们使用生物信息学工具、主成分分析和祖先序列重建，结合蛋白质生物化学，来分析这些酶的自然序列变异。对分选酶超家族的主成分分析区分了先前描述的类别，并识别了每个分选酶家族内结构保守环中序列变异相对较高的区域，包括活性位点附近的区域。使用祖先序列重建，我们确定了祖先葡萄球菌和链球菌 A 类分选酶蛋白的序列。酶测定表明，祖先链球菌酶相对活跃，并且与其他 A 类链球菌分选酶具有相似的序列变异。总而言之，我们强调如何利用自然序列变异来研究这个重要的蛋白质家族，认为这些和类似的技术可用于发现或设计具有增加的催化效率和/或选择性的分选酶，用于分选酶介导的连接实验。
Gram-positive bacteria are some of the earliest known life forms, diverging from gram-negative bacteria 2 billion years ago. These organisms utilize sortase enzymes to attach proteins to their peptidoglycan cell wall, a structural feature that distinguishes the two types of bacteria. The transpeptidase activity of sortases make them an important tool in protein engineering applications, e.g., in sortase-mediated ligations or sortagging. However, due to relatively low catalytic efficiency, there are ongoing efforts to create better sortase variants for these uses. Here, we use bioinformatics tools, principal component analysis and ancestral sequence reconstruction, in combination with protein biochemistry, to analyze natural sequence variation in these enzymes. Principal component analysis on the sortase superfamily distinguishes previously described classes and identifies regions of relatively high sequence variation in structurally-conserved loops within each sortase family, including those near the active site. Using ancestral sequence reconstruction, we determined sequences of ancestral Staphylococcus and Streptococcus Class A sortase proteins. Enzyme assays revealed that the ancestral Streptococcus enzyme is relatively active and shares similar sequence variation with other Class A Streptococcus sortases. Taken together, we highlight how natural sequence variation can be utilized to investigate this important protein family, arguing that these and similar techniques may be used to discover or design sortases with increased catalytic efficiency and/or selectivity for sortase-mediated ligation experiments.