【作者】 李琼；

【导师】 陈宇星； 周丛照；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 LytA存在于多种链球菌中,包括致病菌肺炎链球菌、假肺炎链球菌和轻型链球菌等。由于它在细菌自溶过程中发挥关键作用而得名自溶素。LytA是锚定在细菌细胞壁上的肽聚糖水解酶,能够水解N-乙酰胞壁酸和L-丙氨酸之间的酰胺键,但是其激活机制和发挥生理功能的分子机制仍然不清楚。我们解析了肺炎链球菌LytA的全长结构,分辨率为2.1A。LytA的全长结构为一个飞镖形状的同源二聚体,其相互作用区域位于C末端。每个亚基都包含有一个N端的酰胺酶结构域和一个C端的胆碱结合结构域,其中胆碱结合结构域有6个胆碱结合重复区,包括5个典型的和1个单层的胆碱结合位点。LytA N端的酰胺酶结构域上具有一个Y型的底物结合沟槽,结构分析结合生化实验证明其可能的底物为肺炎链球菌细胞壁的三个分支：糖链、肽链和磷壁酸。另外,点突变结合酶活实验表明二聚化和所有胆碱结合位点被胆碱分子完全占据,才能使LytA具有完全催化活性的构象,进而使其2个酰胺酶结构域维持合适的距离来切割肽聚糖上相距103A的2个乳酰-酰胺键。另外,我们还对肺炎链球菌中的肽聚糖水解酶CbpD进行了结构和功能研究。为了获得上清表达、稳定性好和状态均一的目的蛋白,我们尝试了CbpD的多个截短版本。虽然最终未能解析其结构,但我们的工作会为后续的研究奠定基础。同时,我们构建了多个肽聚糖水解酶基因的敲除株,发现LytA和LytC倾向于水解初生肽聚糖,并且敲除会影响细菌粘附宿主细胞。更多还原

【Abstract】 LytA is responsible for the autolysis of many Streptococcus species, including pathogens such as S. pneumoniae, S. pseudopneumoniae and S. mitis. The major autolysin LytA is also a peptidoglycan hydrolase, which cleaves the lactyl-amide bond between NAM and L-Ala after anchoring on the bacterial cell wall. However, how this major autolysin achieves full activity remains unknown. Here, the full-length structure of the S. pneumoniae LytA dimer is reported at 2.1 A resolution. Each subunit has an N-terminal amidase domain and a C-terminal choline-binding domain consisting of six choline-binding repeats, which form five canonical and one single-layered choline-binding sites. A putative Y-shaped substrate-binding groove in the amidase domain is proposed to accommodate the three branches of pneumococcal cell wall, which contains glycan chains, peptide and teichoic acid chains, as LytA’s bona fide in vivo substrate. Site-directed mutageneses combined with enzymatic activity assays indicate that dimerization and binding to choline are two independent requirements for the autolytic activity of LytA in vivo. Altogether, it is suggested that dimerization and full occupancy of all choline-binding sites through binding to choline-containing TA chains enable LytA to adopt a fully active conformation which allows the amidase domain to cleave two lactyl-amide bonds located about 103 A apart on the peptidoglycan.In addition, I have worked on pneumococcal peptidoglycan hydrolase CbpD. We have tried hardly to get stable and pure protein with multiple truncated versions of CbpD. Unfortunately, we didn’t get the crystal for structural study. Meanwhile, we have knocked out a series of genes encoding peptidoglycan hydrolases (LytA, LytB, DacA, DacB and CbpD) from S. pneumoniae TIGR4 strain, respectively, to investigate their physiological functions. We found that LytA and LytC prefer hydrolyzing the nascent peptidoglycan and their knockout results in the decrease in adhesion to host cells.更多还原