de las Rivas B, García J L, López R, García P
Centro de Investigaciones Biológicas, CSIC, Velázquez, Madrid, Spain.
Microb Drug Resist. 2001 Fall;7(3):213-22. doi: 10.1089/10766290152652756.
A search to identify proteins with high affinity for choline-containing pneumococcal cell walls (choline-binding proteins) has permitted the localization, cloning, sequencing, and overexpression of a gene (pce), coding for a protein (Pce) that liberates phosphorylcholine from purified cell walls of Streptococcus pneumoniae. The pce gene of the pneumococcal strain R6 encodes a protein of 627 amino acids with a predicted Mr of 72,104. Pce can remove a maximum of 20% phosphorylcholine residues from the cell wall teichoic acid. In silico analysis of Pce shows a modular organization of the enzyme where the choline-binding domain, involved in cell wall substrate recognition, and the catalytic domain are located at the carboxy- and amino-terminal moieties of the protein, respectively. Remarkably, a long tail of 85 amino acids follows the carboxy-terminal domain, a structural feature that had not been described for the published choline-binding proteins. The carboxy-terminal moiety of Pce is assembled by 10 repeated motifs, and the protein has also a cleavable signal peptide of 25 amino acids that renders after its cleavage a mature protein of 69,426 Da (602 amino acids). The pce gene has been expressed in Escherichia coli, and Pce was active when assayed on pneumococcal walls. We have also found that the signal peptide of Pce was functional in E. coli. Biochemical analysis suggested that Pce is the teichoic acid phosphorylcholine esterase of S. pneumoniae that had been biochemically characterized previously. Construction of two pce pneumococcal mutants (R6D and M31D) by insertion-duplication mutagenesis revealed that these mutants grew at a doubling-time similar to those of the parental strains of the wild-type R6 and the lytA-mutant M31, respectively. R6D and M31D were morphologically indistinguishable from the parental strains when whole-mounted cells were observed under the electron microscope and exhibited levels of competence for genetic transformation slightly lower than those reported for R6 and M31.
通过搜索来鉴定对含胆碱的肺炎球菌细胞壁具有高亲和力的蛋白质（胆碱结合蛋白），已实现了一个基因（pce）的定位、克隆、测序及过表达，该基因编码一种能从肺炎链球菌纯化细胞壁中释放磷酰胆碱的蛋白质（Pce）。肺炎球菌菌株R6的pce基因编码一个由627个氨基酸组成的蛋白质，预测分子量为72,104。Pce最多可从细胞壁磷壁酸中去除20%的磷酰胆碱残基。对Pce的计算机分析显示该酶具有模块化结构，其中参与细胞壁底物识别的胆碱结合结构域和催化结构域分别位于蛋白质的羧基末端和氨基末端部分。值得注意的是，羧基末端结构域之后有一条由85个氨基酸组成的长尾巴，这是已发表的胆碱结合蛋白中未曾描述过的结构特征。Pce的羧基末端部分由10个重复基序组成，该蛋白质还有一个由25个氨基酸组成的可裂解信号肽，裂解后产生一个分子量为69,426 Da（602个氨基酸）的成熟蛋白质。pce基因已在大肠杆菌中表达，且在对肺炎球菌细胞壁进行检测时Pce具有活性。我们还发现Pce的信号肽在大肠杆菌中具有功能。生化分析表明，Pce是之前已进行过生化特性鉴定的肺炎链球菌的磷壁酸磷酰胆碱酯酶。通过插入 - 重复诱变构建的两个pce肺炎球菌突变体（R6D和M31D）显示，这些突变体的生长倍增时间分别与野生型R6和亲本菌株lytA突变体M31相似。当在电子显微镜下观察整装细胞时，R6D和M31D在形态上与亲本菌株无明显差异，并且它们的遗传转化感受态水平略低于R6和M31报道的水平。
