Role of N-acetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis peptidoglycan metabolism
Role of N-acetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis peptidoglycan metabolism
复制标题
DOI：
10.1074/jbc.m802323200
复制
发表时间：
2008-07-11
影响因子：
4.8
通讯作者：
Arthur, Michel
中科院分区：
文献类型：
--
作者：
Mesnage, Stephane;Chau, Francoise;Arthur, Michel
通过对粪肠球菌提取液中完整的肽聚糖水解酶的鉴定，得到了两种新的水解酶，我们将其命名为AtlB和AtlC。这两种酶都有相似的模块化结构，包括一个中心催化域与两个LysM肽聚糖结合模块融合。通过对纯化的酶产生的肽聚糖片段进行串联质谱分析，AtlB和AtlC显示了N-乙酰胞壁酰胺酶的活性。编码AtlB和AtlC的基因被单独删除或与编码ATLA的基因一起删除，ATLA是一种先前描述的N-乙酰氨基葡萄糖苷酶。在三个突变体中没有检测到自溶活性，表明ATLA、AtlB和AtlC是粪肠球菌主要的水解酶活性。流式细胞仪分析细胞大小分布表明，ATLA缺失导致了长链的形成。因此，ATLA消化隔膜，是细胞分裂后细胞分离所必需的。我们发现AtlB可以作为ATLA的替代品，尽管这种酶在间隔消化方面效率较低。AtlC基因缺失对细胞形态无影响。用N-[C-14]乙酰氨基葡萄糖标记的肽聚糖显示，与模型生物相比，其周转异常缓慢，几乎完全依赖于ATLA和AtlB的联合活性。与ATLA不同的是，atlB和atlC基因位于假定的前驱噬菌体中。由于AtlB和AtlC是在没有细胞裂解或没有产生噬菌体后代的情况下产生的，这些酶可能被粪肠球菌劫持以促进肽聚糖的代谢。
Identification of the full complement of peptidoglycan hydrolases detected by zymogram in Enterococcus faecalis extracts led to the characterization of two novel hydrolases that we named AtlB and AtlC. Both enzymes have a similar modular organization comprising a central catalytic domain fused to two LysM peptidoglycan-binding modules. AtlB and AtlC displayed N-acetylmuramidase activity, as demonstrated by tandem mass spectrometry analyses of peptidoglycan fragments generated by the purified enzymes. The genes encoding AtlB and AtlC were deleted either alone or in combination with the gene encoding AtlA, a previously describedN-acetylglucosaminidase. No autolytic activity was detected in the triple mutant indicating that AtlA, AtlB, and AtlC account for the major hydrolytic activities in E. faecalis. Analysis of cell size distribution by flow cytometry showed that deletion of atlA resulted in the formation of long chains. Thus, AtlA digests the septum and is required for cell separation after cell division. We found that AtlB could act as a surrogate for AtlA, although the enzyme was less efficient at septum digestion. Deletion of atlC had no impact on cell morphology. Labeling of the peptidoglycan with N-[C-14] acetylglucosamine revealed an unusually slow turnover as compared with model organisms, almost completely dependent upon the combined activities of AtlA and AtlB. In contrast to atlA, the atlB and atlC genes are located in putative prophages. Because AtlB and AtlC were produced in the absence of cell lysis or production of phage progeny, these enzymes may have been hijacked by E. faecalis to contribute to peptidoglycan metabolism.