权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

抗生素耐药性的酶学

基本信息

批准号：
7154090
负责人：
RICHARD N ARMSTRONG
金额：
$ 21.48万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-02-01 至 2009-07-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7154090
关键词：
Antibiotic Resistance Antibiotic Therapy Antibiotics Antimicrobial Resistance Appearance Bacillus (bacterium)Bacillus subtilis Binding Biochemical Brucella melitensis Calorimetry Catalysis Characteristics Chemicals Clinic Clostridium botulinum Complex Cysteine Drug Design Drug resistance Electron Nuclear Double Resonance Elements Enzymatic Biochemistry Enzyme Inhibition Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Epoxide hydrolase Evaluation Fosfomycin Freezing Genome Genomics Glutathione Goals Hydration status Investigation Kinetics Listeria monocytogenes Mediating Metals Patient Care Plasmids Proteins Pseudomonas aeruginosa Reaction Relative (related person)Research Research Project Grants Resistance Role Spectrum Analysis Staphylococcus aureus Structure Subgroup Substrate Interaction Sulfhydryl Compounds Techniques Thermodynamics Titrations X-Ray Crystallography base enzyme structure gene cloning infectious disease treatment inhibitor/antagonist metalloenzyme microbial microorganism pathogen stereochemistry three dimensional structure

项目摘要

In the last two decades it has become increasingly clear that the efficacy of antibiotics for the treatment of infectious diseases is in jeopardy due to the common appearance of drug resistant strains of microorganisms. Understanding the mechanisms of antimicrobial resistance is crucial for effective patient care in the clinic and essential for developing strategies to enhance biodefence against intentionally disseminated of pathogens. Fosfomycin is a potent, broad-spectrum antibiotic effective against both Gram-positive and Gram-negative microorganisms. A decade after its introduction plasmid-mediated resistance to fosfomycin was observed in the clinic. Investigations supported by this project have established that the resistance is due to a metalloenzyme (FosA) that catalyzes the addition ofglutathione to the antibiotic, rendering it inactive. Similar resistance elements have now been shown to exist in the genomes of several pathogenic microorganisms including, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus anthrasis, Brucella melitensis, Listeria monocytogenes and Clostridium botulinum. Genomic and biochemical analysis from this project suggest that there are three distinct subgroups ofmetalloenzymes, termed FosA, FosB and FosX, that confer resistance through somewhat different chemical mechanisms. The objectives of this research project are to identify plasmid and genomically encoded proteins involved in microbial resistance to fosfomycin and to elucidate the underlying structural and mechanistic enzymology of resistance. These objectives will be accomplished by integrating enzymological, biophysical and genomic analyses of the resistance problem. The three-dimensional structures of the FosA from Pseudomonas aeruginosa and its relatives FosB and FosX will be determined by X-ray crystallography. The chemical and ldnede mechanisms of catalysis will be elucidated by: (i) examination of the inner coordination sphere of Mn 2+ in FosA and FosX by EPR and ENDOR spectroscopy; (ii) a steady state kinetic analysis of the thiol selectivity of FosA and FosB, and (iii) a mechanistic study of the unique hydration reaction catalyzed by FosX. Potential transition state inhibitors will investigated by structural, spectroscopic and kinetic techniques. The thermodynamics of the interaction of substrates and inhibitors with the enzymes will be examined by isothermal titration calorimetry Particular emphasis will be placed on the enzymes from the pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum. The intent of this investigation is to establish the mechanistic and structural bases for the design of drugs to counter both plasmid borne and genomically encoded resistance to fosfomycin.

在过去的二十年里，人们越来越清楚地认识到抗生素在治疗以下疾病方面的功效：由于耐药微生物菌株的普遍出现，传染病正处于危险之中。了解抗菌药物耐药性的机制对于临床和患者的有效护理至关重要对于制定加强针对故意传播病原体的生物防御的战略至关重要。磷霉素是一种强效广谱抗生素，对革兰氏阳性菌和革兰氏阴性菌均有效微生物。在其引入十年后，在细菌中观察到质粒介导的磷霉素耐药性诊所。该项目支持的调查已确定该耐药性是由金属酶引起的（FosA）催化谷胱甘肽添加到抗生素中，使其失去活性。类似电阻元件现在已被证明存在于几种病原微生物的基因组中，包括假单胞菌铜绿假单胞菌、金黄色葡萄球菌、炭疽杆菌、羊布鲁氏菌、单核细胞增生李斯特菌和肉毒杆菌。该项目的基因组和生化分析表明存在三种不同的金属酶的亚类，称为 FosA、FosB 和 FosX，通过有些不同的方式赋予抗性化学机制。该研究项目的目标是鉴定质粒和基因组编码参与微生物对磷霉素耐药性的蛋白质并阐明潜在的结构和机制抗性酶学。这些目标将通过整合酶学、生物物理和耐药问题的基因组分析。假单胞菌 FosA 的三维结构铜绿假单胞菌及其近亲 FosB 和 FosX 将通过 X 射线晶体学测定。化学和催化机制将通过以下方式阐明： (i) 检查 Mn 2+ 的内部配位层通过 EPR 和 ENDOR 光谱分析 FosA 和 FosX； (ii) 硫醇选择性的稳态动力学分析 FosA 和 FosB，以及 (iii) FosX 催化的独特水合反应的机理研究。潜在的过渡态抑制剂将通过结构、光谱和动力学技术进行研究。这底物和抑制剂与酶相互作用的热力学将通过等温进行检查滴定量热法特别强调来自病原体假单胞菌的酶铜绿假单胞菌、金黄色葡萄球菌、单核细胞增生李斯特菌和肉毒杆菌。这样做的意图研究的目的是为设计对抗质粒的药物建立机制和结构基础遗传性和基因组编码的磷霉素抗性。