CHARACTERIZATION OF LPXTGASE FROMSTAPHYLOCOCCUS AUREUS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细菌表面蛋白是病原体感染和存活的重要分子。革兰氏阳性细菌上的表面蛋白已显示通过转肽酶（称为分选酶）附着，所述转肽酶切割在这些细菌上几乎所有表面蛋白的C末端附近发现的LPXTG序列。我们以前确定了一种独特的酶（LPXTGase）从化脓性链球菌，也切割LPXTG基序的催化活性高于分选酶，这表明它在附着过程中发挥了重要作用。我们现在已经从金黄色葡萄球菌中纯化并表征了LPXTGase，并且发现它与S.化脓酶色葡萄金黄色葡萄球菌的酶是糖基化的，并含有不寻常的氨基酸，像它的链球菌对应物。与链球菌酶一样，葡萄球菌LPXTGase具有在肽聚糖中发现的过量氨基酸，即，谷氨酰胺/谷氨酸、甘氨酸、丙氨酸和赖氨酸，此外，我们发现这些氨基酸以与它们在相应生物体的肽聚糖中发现的比例完全相同的比例存在于酶中。这表明负责壁组装的酶也可能在细胞壁的构建中发挥作用。 LPXTGase。 在目前的工作中，我们正在寻求通过质谱法对这些不寻常的酶进行测序。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacterial surface proteins are important molecules in the infectivity and survival of pathogens. Surface proteins on gram-positive bacteria have been shown to attach via a transpeptidase, termed sortase, that cleaves an LPXTG sequence found close to the C termini of nearly all surface proteins on these bacteria. We previously identified a unique enzyme (LPXTGase) from Streptococcus pyogenes that also cleaves the LPXTG motif with a catalytic activity higher than that of sortase, suggesting that it plays an important role in the attachment process. We have now purified and characterized an LPXTGase from Staphylococcus aureus and found that it has both similar and unique features compared to the S. pyogenes enzyme. The S. aureus enzyme is glycosylated and contains unusual amino acids, like its streptococcal counterpart. Like the streptococcal enzyme, staphylococcal LPXTGase has an overrepresentation of amino acids found in the peptidoglycan, i.e., glutamine/glutamic acid, glycine, alanine, and lysine, and furthermore, we find that these amino acids are present in the enzyme at precisely the same ratio at which they are found in the peptidoglycan for the respective organism. This suggests that enzymes responsible for wall assembly may also play a role in the construction of LPXTGase. In the present work, we are seeking to sequence these unusual enzymes by mass spectrometry.