| 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. |
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