ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

抗生素耐药性的酶学

• 批准号: 6678647
• 负责人: RICHARD N ARMSTRONG
• 金额: $ 13.83万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678647
• 关键词: Clostridium botulinum; Listeria; Pseudomonas aeruginosa; Staphylococcus aureus; X ray crystallography; antibiotics; apoenzymes; calorimetry; chemical structure function; divalent cations; drug resistance; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; metalloenzyme; stereochemistry

DESCRIPTION (provided by applicant): 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 biodefense 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 of glutathione 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 of metalloenzymes, 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 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.

描述（由申请人提供）： 在过去的二十年中，越来越清楚的是，由于微生物的耐药菌株的普遍出现，抗生素用于治疗感染性疾病的功效处于危险之中。了解抗菌素耐药性的机制对于临床有效的患者护理至关重要，对于制定战略以加强对故意传播的病原体的生物防御至关重要。磷霉素是一种有效的广谱抗生素，对革兰氏阳性和革兰氏阴性微生物都有效。在其引入十年后，在临床上观察到质粒介导的对磷霉素的耐药性。由该项目支持的研究已经确定，耐药性是由于金属酶（FosA）催化谷胱甘肽加入抗生素，使其失活。类似的抗性元件现已显示存在于几种病原微生物的基因组中，包括铜绿假单胞菌、金黄色葡萄球菌、炭疽杆菌、羊种布鲁氏菌、单核细胞增生李斯特菌和肉毒梭菌。该项目的基因组和生物化学分析表明，有三种不同的金属酶亚组，称为FosA，FosB和FosX，通过不同的化学机制赋予抗性。本研究项目的目的是确定质粒和基因组编码的蛋白质参与微生物耐磷霉素，并阐明潜在的结构和机制的耐药酶学。这些目标将通过整合抗性问题的酶学、生物物理和基因组分析来实现。来自铜绿假单胞菌的FosA及其亲属FosB和FosX的三维结构将通过X射线晶体学确定。催化的化学机理将阐明：（i）检查的内配位球的Mn 2+在FosA和FosX的EPR和ENDOR光谱;（ii）的硫醇选择性的FosA和FosB的稳态动力学分析，和（iii）由FosX催化的独特的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。底物和抑制剂与酶的相互作用的热力学将通过等温滴定量热法进行检查。特别强调的是来自病原体铜绿假单胞菌、金黄色葡萄球菌、单核细胞增生李斯特菌和肉毒梭菌的酶。这项研究的目的是为设计药物建立机制和结构基础，以对抗质粒携带的和基因组编码的磷霉素耐药性。