Investigating Substrate Specificity of Sortases

描述(申请人提供)：革兰氏阳性细菌是许多医院感染和疾病的病原体，包括骨髓炎和肺炎。大多数革兰氏阳性细菌都含有分类酶，这是一种转肽酶，可以识别蛋白质C末端附近的短肽序列。含有这种识别基序的蛋白质随后被锚定到肽聚糖上，并需要通过作为粘附素或通过中介逃避宿主的免疫系统来确定感染。一个这样的分类酶，srtA，在体外识别和处理序列Leu-Pro-X-Thr-Gly基序。生物信息学分析预测，SrtA可能识别LPXTG变体以及第六个氨基酸。另一个分类酶家族，亚家族-3，也被预测处理类似的识别基序。我们建议确定这些分类酶的底物特异性，以了解体内识别。我们还将使用核磁共振波谱来解析山梨酸酶-底物复合体的结构。该项目的结果将提供这些转肽酶如何发挥作用的详细信息，并将确定参与识别的残基。这项工作将补充正在进行的索酸酶抑制剂研究。

DESCRIPTION (provided by applicant): Gram-positive bacteria are the causative agents of many nosocomial infections and diseases including osteomyelitis and pneumonia. The majority of Gram-positive bacteria harbor sortases, which are transpeptidases that recognize a short peptide sequence found near the C-terminus of proteins. Proteins harboring this recognition motif are subsequently anchored to the peptidoglycan and are required to establish infection by acting as adhesins or by mediating evasion from the host's immune system. One such sortase, SrtA, recognizes and processes the sequence, Leu-Pro-X-Thr-Gly motif in vitro. Bioinformatics analyses predict SrtA may recognize LPXTG variants as well as a 6th amino acid. Another family of sortases, subfamily-3, is also predicted to process similar recognition motifs. We propose to determine the substrate specificity of these sortases to understand in vivo recognition. We will also use NMR spectroscopy to solve the structures of the sortase-substrate complexes. Results from this project will afford details on how these transpeptidases function and will identify residues participating in recognition. This work will complement ongoing sortase inhibitor studies.