Mechanism and Inhibition of Bacterial Transglycosylases and Transpeptidases.

细菌转糖基酶和转肽酶的机制和抑制。

• 批准号: 8664874
• 负责人: Suzanne Walker
• 金额: $ 53.39万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-11 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8664874
• 关键词: Accounting; Address; Affect; Alanine; Anabolism; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Bambermycins; Binding; Biochemical; Biological; Biological Assay; Biological Factors; Carboxypeptidase; Catalysis; Cell Wall; Cell physiology; Clinical; Combined Modality Therapy; Complex; Defect; Disaccharides; Endopeptidases; Enterococcus faecalis; Enzymatic Biochemistry; Enzymes; Escherichia coli; Family; Foundations; Genes; Glycine; Health; Human; Hydrolysis; In Vitro; Infection; Knowledge; Lead; Length; Lipids; Measures; Methicillin Resistance; Methods; Modeling; Molecular; Molecular Weight; Monitor; Multienzyme Complexes; Organism; Pathway interactions; Penicillin-Binding Proteins; Peptide Synthesis; Peptides; Peptidoglycan; Peptidoglycan glycosyltransferase; Peptidyltransferase; Phase; Polymers; Polysaccharides; Proteins; Public Health; Reaction; Reporting; Research; Research Personnel; Resistance; Roentgen Rays; Role; Staging; Staphylococcus aureus; Structure; Substrate Specificity; Testing; Variant; Work; analog; base; beta-Lactam Resistance; beta-Lactams; chemical reaction; chemical synthesis; crosslink; design; enzyme model; genetic regulatory protein; inhibitor/antagonist; insight; methicillin resistant Staphylococcus aureus; novel strategies; polymerization; preference; research study; screening; stem; structural biology; tool; transpeptidation; virtual

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.

描述(申请人提供)：抗药性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。许多临床上使用的抗生素针对的是肽聚糖(PG)生物合成的最后几步，这包括由肽聚糖糖基转移酶(PGTS)聚合二糖-多肽亚基和通过转肽酶(TPS)使聚合的链交联。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PGTS和TPS的功能，人们必须能够制作复杂的底物，以区分连接相同分子的不同酶亚基。我们提出了涉及使用肽聚糖片段的三个具体目标，以解决关于PGTS和TPS知识的主要差距。例如，尽管TPS是β-内酰胺类药物的致命靶标，但它们几乎完全没有特征性。在目标I中，我们建议a)鉴定含有封闭的非还原末端的四糖底物，该末端激活PGT用于延伸，以及b)使用这些分子来获得PGT“延伸复合体”的晶体结构。我们和其他人以前用莫诺霉素结晶的PGT结构域将用于这些研究。具有伸长活性的PGT：底物复合体的结构将为催化作用提供新的见解，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)使肽聚糖聚合物底物能够活化但不能交联，以及b)将它们与能够活化和交联的聚合物底物结合使用来开发报告细菌转肽酶对多肽的活化、水解和交联性的检测方法。大肠杆菌PBP1a和粪肠球菌PBP2a将被用作这些实验的模型酶。检测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的TPS的底物特异性成为可能。在目标III中，我们建议a)制作金黄色葡萄球菌Lipid II的三个主茎多肽变体，并使用这些底物来制备相应的PG聚合物；以及b)表征MRSA中对β-内酰胺敏感和β-内酰胺耐药的转肽激活、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与干肽分支有关的基因可以恢复对含有内在耐药转肽酶的MRSA菌株的β-内酰胺敏感性。没有生化证据支持这一假设，因为金黄色葡萄球菌TPS的底物偏好还没有被研究过。AIM III的实验结果为克服MRSA的新方法提供了启示，这种方法涉及将β-内酰胺类化合物与靶向参与甲氧西林耐药的其他蛋白质的化合物结合起来。