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: 8478130
• 负责人: Suzanne Walker
• 金额: $ 51.51万
• 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 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8478130
• 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（由申请人提供）：抗生素耐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靶向肽聚糖（PG）生物合成的最后步骤，其涉及肽聚糖糖基转移酶（PGT）聚合二硫代戊二酰亚胺-肽亚基以及转肽酶（TP）交联聚合链。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基质由单一二糖底物组装成复杂的三维聚合物。为了了解PGTs和TP的功能，必须能够制造复杂的底物，以区分与相同分子偶联的酶的不同亚位点。我们提出了三个具体的目标，涉及使用肽聚糖片段，以解决有关PGTs和TP的知识的主要差距。例如，虽然TP是β-内酰胺类药物的致死靶点，但它们几乎完全没有特征。在目的I中，我们提出a）鉴定含有封闭的非还原末端的四糖底物，其活化PGTs用于延伸，和B）使用这些分子获得PGT“延伸复合物”的晶体结构。我们和其他人以前用默诺霉素结晶的PGT结构域将用于这些研究。一个结构的延伸主管PGT：底物复合物将提供新的见解催化和虚拟筛选和设计的抑制剂的新基础。在目标II中，我们提出a）制备能够活化但不交联的肽聚糖聚合物底物，和B）将它们与能够活化和交联的聚合物底物结合使用，以开发报告细菌转肽酶对肽的活化、水解和交联的测定。E. coli PBP 1A和E.将使用粪杆菌PBP 2A作为这些实验的模型酶。测定TP活性的能力将使得有可能解决TP调节蛋白在细菌中的功能，并表征来自其他生物体的TP的底物特异性。在目的III中，我们提出a）制备S.金黄色葡萄球菌脂质II，并使用这些底物制备相应的PG聚合物;和B）表征MRSA中β-内酰胺敏感性和β-内酰胺抗性转肽活化、水解和交联这些聚合物的能力。有人提出S.金黄色葡萄球菌TP具有不同的底物偏好，并且这解释了为什么删除参与茎肽分支的基因恢复了对含有内在抗性转肽酶的MRSA菌株的β-内酰胺敏感性。目前还没有生物化学证据支持这一假说，因为S.金黄色葡萄球菌TP尚未被检测。目标III中的实验结果对克服MRSA的新方法具有影响，该方法涉及将β-内酰胺与靶向与甲氧西林耐药性有关的其他蛋白质的化合物相结合。