Peptidoglycan is a vital component of the bacterial cell wall, and its dynamic remodeling by NlpC/p60 hydrolases is crucial for proper cell division and survival. Beyond these essential functions, we previously discovered that Enterococcus species express and secrete the NlpC/p60 hydrolase-secreted antigen A (SagA), whose catalytic activity can modulate host immune responses in animal models. However, the localization and peptidoglycan hydrolase activity of SagA in Enterococcus was still unclear. In this study, we show that SagA contributes to a triseptal structure in dividing cells of enterococci and localizes to sites of cell division through its N-terminal coiled-coil domain. Using molecular modeling and site-directed mutagenesis, we identify amino acid residues within the SagA-NlpC/p60 domain that are crucial for catalytic activity and potential substrate binding. Notably, these studies revealed that SagA may function via a catalytic Cys-His dyad instead of the predicted Cys-His-His triad, which is conserved in SagA orthologs from other Enterococcus species. Our results provide key additional insight into peptidoglycan remodeling in Enterococcus by SagA NlpC/p60 hydrolases.

中文翻译：

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肠球菌 NlpC/p60 肽聚糖水解酶 SagA 定位于细胞分裂位点，蛋白酶活性仅需要催化二元体

肽聚糖是细菌细胞壁的重要组成部分，其通过 NlpC/p60 水解酶进行的动态重塑对于正常的细胞分裂和存活至关重要。除了这些基本功能之外，我们之前发现肠球菌属表达和分泌 NlpC/p60 水解酶分泌抗原 A (SagA)，其催化活性可以调节动物模型中的宿主免疫反应。然而，SagA 在肠球菌中的定位和肽聚糖水解酶活性仍然不清楚。在这项研究中，我们表明 SagA 有助于肠球菌分裂细胞的三隔膜结构，并通过其 N 末端卷曲螺旋结构域定位到细胞分裂位点。使用分子建模和定点诱变，我们确定了 SagA-NlpC/p60 结构域中对催化活性和潜在底物结合至关重要的氨基酸残基。值得注意的是，这些研究表明，SagA 可能通过催化 Cys-His 二联体而不是预测的 Cys-His-His 三联体发挥作用，后者在其他肠球菌属的 SagA 直系同源物中是保守的。我们的结果为SagA NlpC/p60 水解酶对肠球菌中的肽聚糖重塑提供了关键的额外见解。