Sáiz José L, López-Zumel Consuelo, Monterroso Begoña, Varea Julio, Arrondo José Luis R, Iloro Ibon, García José L, Laynez José, Menéndez Margarita
Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
Protein Sci. 2002 Jul;11(7):1788-99. doi: 10.1110/ps.4680102.
The Ejl amidase is coded by Ej-1, a temperate phage isolated from the atypical pneumococcus strain 101/87. Like all the pneumococcal cell-wall lysins, Ejl has a bimodular organization; the catalytic region is located in the N-terminal module, and the C-terminal module attaches the enzyme to the choline residues of the pneumococcal cell wall. The structural features of the Ejl amidase, its interaction with choline, and the structural changes accompanying the ligand binding have been characterized by CD and IR spectroscopies, differential scanning calorimetry, analytical ultracentrifugation, and FPLC. According to prediction and spectroscopic (CD and IR) results, Ejl would be composed of short beta-strands (ca. 36%) connected by long loops (ca. 17%), presenting only two well-predicted alpha-helices (ca. 12%) in the catalytic module. Its polypeptide chain folds into two cooperative domains, corresponding to the N- and C-terminal modules, and exhibits a monomer <--> dimer self-association equilibrium. Choline binding induces small rearrangements in Ejl secondary structure but enhances the amidase self-association by preferential binding to Ejl dimers and tetramers. Comparison of LytA, the major pneumococcal amidase, with Ejl shows that the sequence differences (15% divergence) strongly influence the amidase stability, the organization of the catalytic module in cooperative domains, and the self-association state induced by choline. Moreover, the ligand affinity for the choline-binding locus involved in regulation of the amidase dimerization is reduced by a factor of 10 in Ejl. Present results evidence that sequence differences resulting from the natural variability found in the cell wall amidases coded by pneumococcus and its bacteriophages may significantly alter the protein structure and its attachment to the cell wall.
Ejl酰胺酶由Ej-1编码，Ej-1是从非典型肺炎球菌菌株101/87中分离出的一种温和噬菌体。与所有肺炎球菌细胞壁溶素一样，Ejl具有双模块结构；催化区域位于N端模块，C端模块将酶附着于肺炎球菌细胞壁的胆碱残基上。Ejl酰胺酶的结构特征、其与胆碱的相互作用以及配体结合时伴随的结构变化已通过圆二色光谱和红外光谱、差示扫描量热法、分析超速离心和快速蛋白质液相色谱进行了表征。根据预测和光谱（圆二色光谱和红外光谱）结果，Ejl由短β链（约36%）通过长环（约17%）连接而成，在催化模块中仅呈现两个预测良好的α螺旋（约12%）。其多肽链折叠成两个协同结构域，分别对应于N端和C端模块，并呈现单体<-->二聚体自缔合平衡。胆碱结合在Ejl二级结构中诱导小的重排，但通过优先结合Ejl二聚体和四聚体增强酰胺酶的自缔合。主要肺炎球菌酰胺酶LytA与Ejl的比较表明，序列差异（15%的分歧）强烈影响酰胺酶的稳定性、催化模块在协同结构域中的组织以及胆碱诱导的自缔合状态。此外，Ejl中参与酰胺酶二聚化调节的胆碱结合位点的配体亲和力降低了10倍。目前的结果证明，肺炎球菌及其噬菌体编码的细胞壁酰胺酶中自然变异导致的序列差异可能会显著改变蛋白质结构及其与细胞壁的附着。
