CHARACTERIZATION OF LPXTGASE FROMSTAPHYLOCOCCUS AUREUS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细菌表面蛋白是影响病原体感染性和生存的重要分子。革兰氏阳性细菌上的表面蛋白被证明是通过一种名为Sortase的转肽酶附着的，这种酶能切割一段LPXTG序列，该序列靠近这些细菌上几乎所有表面蛋白的C末端。我们以前从化脓性链球菌中发现了一种独特的酶(LPXTGase)，它也能裂解LPXTG基序，其催化活性高于Sortase，这表明它在附着过程中发挥着重要作用。我们现在已经从金黄色葡萄球菌中纯化并鉴定了一种LPXTGase，发现它与化脓性葡萄球菌的酶具有相似和独特的特性。金黄色葡萄球菌的酶是糖基化的，含有不同寻常的氨基酸，就像链球菌的酶一样。与链球菌酶一样，葡萄球菌LPXTGase也具有在肽聚糖中发现的氨基酸的过度表达，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在酶中的存在比例与它们在相应生物体的肽聚糖中的比例完全相同。这表明，负责壁面组装的酶可能也在构建 LPXTGase。 在目前的工作中，我们正在寻求通过质谱学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.