Mechanism and Inhibition of Bacterial Transglycosylases and Transpeptidases.

描述（由申请人提供）：抗生素耐药细菌感染对人类健康构成严重威胁，迫切需要克服这些感染的策略。许多临床使用的抗生素靶向肽聚糖（PG）生物合成的最后步骤，包括肽聚糖糖基转移酶（PGTs）对双糖肽亚基的聚合和转肽酶（TPs）对聚合链的交联。我们对这些步骤的理解存在重大差距，这阻碍了开发新抗生素的努力。PG基质由单一的双糖底物组装成复杂的三维聚合物。为了了解pgt和TPs的功能，人们必须能够设计复杂的底物来区分配对相同分子的酶的不同亚位。我们提出了三个具体目标，涉及使用肽聚糖片段来解决关于pgt和tp知识的主要空白。例如，虽然TPs是β -内酰胺的致命目标，但它们几乎完全没有被表征。在Aim I中，我们建议a)鉴定含有阻断非还原端的四糖底物，激活PGT的延伸，b)使用这些分子获得PGT“延伸复合物”的晶体结构。我们和其他人之前用莫诺霉素结晶的PGT结构域将用于这些研究。延伸能力强的PGT：底物复合物的结构将为催化研究提供新的见解，并为抑制剂的虚拟筛选和设计提供新的基础。在Aim II中，我们建议a)制造能够激活但不能交联的肽聚糖聚合物底物，b)将它们与能够激活和交联的聚合物底物结合使用，以开发报告细菌转肽酶的肽激活，水解和交联的分析。大肠杆菌PBP1A和粪肠杆菌PBP2A将作为这些实验的模型酶。测定TP活性的能力将使解决细菌中TP调节蛋白的功能和表征来自其他生物体的TP的底物特异性成为可能。在Aim III中，我们建议a)制备金黄色葡萄球菌脂质II的三个主要茎肽变体，并利用这些底物制备相应的PG聚合物；b)表征MRSA中β -内酰胺敏感和β -内酰胺耐药转肽类激活、水解和交联这些聚合物的能力。有人提出金黄色葡萄球菌TPs具有不同的底物偏好，这解释了为什么删除涉及茎肽分支的基因可以恢复对含有内在抗性转肽酶的MRSA菌株的β -内酰胺敏感性。由于金黄色葡萄球菌TPs对底物的偏好尚未得到检验，因此尚无生物化学证据支持这一假设。Aim III的实验结果对克服MRSA的新方法具有启示意义，这些新方法涉及将β -内酰胺与靶向甲氧西林耐药的其他蛋白质的化合物结合。

DESCRIPTION (provided by applicant): Antibiotic resistant bacterial infections pose a serious threat to human health and strategies to overcome these infections are desperately needed. Many clinically used antibiotics target the final steps of peptidoglycan (PG) biosynthesis, which involve the polymerization of disaccharide-peptide subunits by peptidoglycan glycosyltransferases (PGTs) and the crosslinking of the polymerized chains by transpeptidases (TPs). There are major gaps in our understanding of these steps, which has hampered efforts to develop new antibiotics. The PG matrix is assembled into a complex three-dimensional polymer from a single disaccharide substrate. In order to understand how the PGTs and TPs function, one must be able to make complicated substrates designed to discriminate between different subsites of enzymes that couple identical molecules. We propose three specific aims involving the use of peptidoglycan fragments to address major gaps in knowledge about PGTs and TPs. For example, although the TPs are the lethal targets of the beta-lactams, they remain almost completely uncharacterized. In Aim I we propose to a) identify tetrasaccharide substrates containing a blocked non-reducing end that activate PGTs for elongation, and b) to use these molecules to obtain a crystal structure of the PGT "elongation complex". PGT domains we and others have previously crystallized with moenomycin will be used for these studies. A structure of an elongation competent PGT:substrate complex would provide new insights into catalysis and a new basis for virtual screening and design of inhibitors. In Aim II, we propose to a) make peptidoglycan polymer substrates capable of activation but not crosslinking, and b) to use them in conjunction with polymer substrates capable of activation and crosslinking to develop assays that report on peptide activation, hydrolysis, and crosslinking by bacterial transpeptidases. E. coli PBP1A and E. faecalis PBP2A will be used as model enzymes for these experiments. The ability to assay TP activity will make it possible to address the functions of TP-regulatory proteins in bacteria and to characterize the substrate specificities of TPs from other organisms. In Aim III, we propose to a) make the three main stem-peptide variants of S. aureus Lipid II and use these substrates to make the corresponding PG polymers; and b) to characterize the abilities of the beta-lactam sensitive and beta-lactam resistant transpeptides in MRSA to activate, hydrolyze, and crosslink these polymers. It has been proposed that the S. aureus TPs have different substrate preferences, and that this explains why deleting genes involved in stem peptide branching restores beta-lactam sensitivity to MRSA strains containing an intrinsically resistant transpeptidase. There is no biochemical evidence for this hypothesis since the substrate preferences of the S. aureus TPs have not been examined. The results of the experiments in Aim III have implications for new approaches to overcome MRSA that involve combining a beta-lactam with compounds that target other proteins involved in methicillin resistance.