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，由于微生物的耐药性菌株的共同出现，抗生素治疗传染病的疗效受到危险。了解抗菌素耐药性的机制对于诊所中的有效患者护理至关重要，对于制定策略以增强有意传播病原体的策略至关重要。 Fosfomycin是一种有效的，宽光谱的抗生素，可对革兰氏阳性和革兰氏阴性微生物有效。在引入质粒介导的fosfomycin的抗性十年后，在诊所观察到了对fosfomycin的抗性。该项目支持的研究表明，抗性是由于金属酶（FOSA）催化在抗生素中添加谷胱甘肽，从而使其不活跃。现在已经证明，类似的抗性元件存在于几种致病微生物的基因组中，包括铜绿假单胞菌，金黄色葡萄球菌，炭疽芽孢杆菌，布鲁氏菌，布鲁氏菌，李斯特氏菌，单核细胞增生李斯特氏菌和肉豆蔻菌。该项目的基因组和生化分析表明，有三个不同的亚组，称为FOSA，FOSB和FOSX，它们通过某种不同的化学机制赋予了抗性。该研究项目的目标是鉴定质粒和基因组编码的蛋白质，涉及对小霉素的微生物耐药性，并阐明耐药性的基本结构和机械酶学。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。铜绿假单胞菌及其亲戚fosb和fosx的FOSA的三维结构将由X射线晶体学确定。催化的化学机制将通过以下方式阐明：（i）通过EPR和Endor光谱检查在FOSA和FOSX中MN 2+的内部配位球体； （ii）对FOSA和FOSB的硫醇选择性的稳态动力学分析，以及（iii）对由FOSX催化的独特水合反应的机械研究。潜在的过渡状态抑制剂将通过结构，光谱和动力学技术研究。将通过等温滴定量热法检查底物和抑制剂与酶的相互作用的热力学将特别强调，这将放在病原体铜绿假单胞菌，葡萄球菌，金黄色葡萄球菌，单细胞基因的listeria listeria andocytogenes and Clostrtrinim bottrinum botlinum bottrinum上。这项研究的目的是建立用于设计药物的机械和结构碱基，以应对质粒传播的质粒和基因组编码对fosfymycin的抗性。