Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany.
Microbial Genetics, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany, and.
J Biol Chem. 2014 Apr 18;289(16):11083-11094. doi: 10.1074/jbc.M114.557306. Epub 2014 Mar 5.
The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.
金黄色葡萄球菌的双功能主要自溶素 AtlA 在细胞分裂过程中在两个不同的位点切割细菌的肽聚糖网络（PGN）。该酶的缺失导致细胞簇较大且分裂模式紊乱，表明 AtlA 可能是开发新型抗生素的有前途的靶标。 AtlA 的两个功能之一由 N-乙酰基muramyl-L-丙氨酸酰胺酶 AmiA 执行，该酶切割 PGN 中碳水化合物和肽部分之间的键。为了确定 PGN 识别的结构要求和裂解的酶促机制，我们以 1.55 Å 的分辨率解决了与肽聚糖衍生配体复合的 AmiA 催化结构域（AmiA-cat）的晶体结构。结构中清楚地显示了肽主干，通过对接进入细长的凹槽与蛋白质残基形成广泛的接触。电子密度不太明确，并且表面特征的分析表明碳水化合物主链和五肽桥的可能位置。底物特异性分析支持五肽桥对于适合 AmiA-cat 的结合裂缝的重要性。通过五肽桥将 l-赖氨酸与 d-丙氨酸连接的金黄色葡萄球菌的 PGN 完全水解，而通过五肽桥将 meso-二氨基庚二酸直接与 d-丙氨酸连接的枯草芽孢杆菌的 PGN 则不被水解。 AmiA-cat 的活性位点突变体 H370A 完全失活，进一步支持了 AmiA 的提议的催化机制。报告的结构不仅是第一个存在于进行亲核攻击的 PGN 成分和水的任何细菌酰胺酶中，而且也是第一个重要的人类病原体的肽聚糖酰胺酶复合物结构。
