Activity of the major staphylococcal autolysin Atl
Activity of the major staphylococcal autolysin Atl
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DOI：
10.1111/j.1574-6968.2006.00281.x
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发表时间：
2006-06-01
影响因子：
2.1
通讯作者：
Goetz, Friedrich
中科院分区：
文献类型：
--
作者：
Biswas, Raja;Voggu, Lalitha;Goetz, Friedrich
金黄色葡萄球菌(ATLA)和表皮葡萄球菌(ATIL)的主要自溶酶都是研究较多的酶。在这里，我们在金黄色葡萄球菌中创建了一个ATLA缺失突变体，它形成了大的细胞团，并且是生物被膜阴性的。在电子显微镜下，突变型细胞由粗糙的细胞外表面区分开来。该突变体可以利用表皮葡萄球菌的ATLE基因进行补充。为了研究ATLE重复序列的作用，我们在大肠杆菌中表达了该基因编码的氨基酶结构域，不含重复区域(AMIE)或两个重复区域(AMIE-R-1，R-2)，或仅含三个重复区域(R-1，R-2，R-3)作为N-末端His-Tag融合蛋白。AMIE-R-1、R-2与AMIE-R-1、R-2的细胞壁裂解活性略有差异。这些重复序列与分离的肽聚糖具有良好的结合亲和力，可能有助于将酰胺酶靶向底物。AMIE和AMIE-R-1、R-2具有广泛的底物专一性，与缺乏壁磷壁酸和/或O-乙酰化的肽聚糖的活性相似。由于ATLA和ATIL的酰胺酶活性尚未得到生化证明，我们使用纯化的AMIE-R-1、R-2来确定准确的肽聚糖裂解位点。我们提供了第一个证据，证明酰胺酶确实断裂了N-乙酰胞壁酸和L-丙氨酸之间的酰胺键。
The major autolysin of Staphylococcus aureus (AtlA) and of Staphylococcus epidermidis (AtlE) are well-studied enzymes. Here we created an atlA deletion mutant in S. aureus that formed large cell clusters and was biofilm-negative. In electron micrographs, the mutant cells were distinguished by rough outer cell surface. The mutant could be complemented using the atlE gene from S. epidermidis. To study the role of the repetitive sequences of atlE, we expressed in Escherichia coli the amidase domain encoded by the gene, carrying no repeat regions (amiE) or two repeat regions (amiE-R-1,R-2), or the three repeat regions alone (R-1,R-2,R-3) as N-terminal His-tag fusion proteins. Only slight differences in the cell wall lytic activity between AmiE and AmiE-R-1,R-2 were observed. The repetitive sequences exhibit a good binding affinity to isolated peptidoglycan and might contribute to the targeting of the amidase to the substrate. AmiE and AmiE-R-1,R-2 have a broad substrate specificity as shown by similar activities with peptidoglycan lacking wall teichoic acid, O-acetylation, or both. As the amidase activity of AtlA and AtlE has not been proved biochemically, we used purified AmiE-R-1,R-2 to determine the exact peptidoglycan cleavage site. We provide the first evidence that the amidase indeed cleaves the amide bond between N-acetyl muramic acid and L-alanine.