NMR studies of the enzyme PMM/PGM from P. aeruginosa

铜绿假单胞菌 PMM/PGM 酶的 NMR 研究

• 批准号: 7846568
• 负责人: LESA J BEAMER
• 金额: $ 1.85万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846568
• 关键词: Address; Alginates; Anabolism; Antibiotic Resistance; Bacteria; Biological; Burn injury; Cancer Patient; Catalysis; Collection; Complement; Cyclophilins; Enzymes; Equilibrium; Family; Family member; Frequencies; Goals; Gram-Negative Bacteria; Human; Infection; Investigation; Laboratories; Ligand Binding; Lipopolysaccharides; Metabolism; Methods; Microbial Biofilms; Motion; Nature; Nosocomial Infections; Nuclear Magnetic Resonance; Organism; Pancreatic ribonuclease; Pathway interactions; Phosphoglucomutase; Phosphomannomutase; Play; Protein Dynamics; Proteins; Pseudomonas aeruginosa; Reaction; Relaxation; Role; Testing; Time; Vertebral column; Virulence; Virulence Factors; Work; X-Ray Crystallography; base; carbohydrate metabolism; cystic fibrosis patients; design; enzyme mechanism; inhibitor/antagonist; insight; member; millisecond; mortality; novel; pathogen; research study; rhamnolipid

DESCRIPTION (provided by applicant): Enzymes in the a-D-phosphohexomutase superfamily play critical roles in carbohydrate metabolism and other biosynthetic pathways. The biological importance of these proteins is demonstrated by their distribution in nature: at least one family member appears to be found in all organisms from bacteria to humans. In this proposal, we will characterize novel aspects of one protein in this enzyme family, phosphomannomutase/phosphoglucomutase (PMM/PGM) from P. aeruginosa. This bacterium is an opportunistic human pathogen that often infects cystic fibrosis patients, burn victims, and cancer patients. PMM/PGM participates in the biosynthesis of multiple bacterial exoproducts, including lipopolysaccharide, alginate and rhamnolipid, which are associated with biofilm formation and increased virulence in P. aeruginosa infections. Because of its key role in the biosynthetic pathways of virulence factors, PMM/PGM is a target for inhibitor design. Previous work has shown that conformational change of this enzyme is required at several key points in its multi-step reaction, which entails two consecutive phosphoryl transfer reactions with an intervening reorientation of the reaction intermediate. Recently, other laboratories have shown evidence for a new paradigm of enzyme dynamics in catalysis: intrinsic fluctuations appear to couple to catalysis. We will test this paradigm in the processive enzyme PMM/PGM. We will use NMR relaxation dispersion to characterize the dynamics of substrate-free (apo) PMM/PGM that occur on a catalytically relevant timescale. In addition, we will initiate investigation of its dynamics during its reversible catalytic reaction. We seek to determine the relationship between protein conformational change, intrinsic fluctuations occurring within milliseconds, and function for this member of the a-D- phosphohexomutase enzyme superfamily. This will aid the long-term goal of developing novel inhibitors that can act at different points in its multi-step reaction. The work in this proposal will initiate characterization of the dynamics of a key enzyme (PMM/PGM) from the bacterial pathogen Pseudomonas aeruginosa. P. aeruginosa infections are the primary cause of mortality for cystic fibrosis patients, and this organism is also a leading cause of hospital-acquired infections. Dynamics studies of PMM/PGM will complement existing structural and mechanistic characterization, with the goal of designing specific inhibitors with utility for the treatment of P. aeruginosa infections.

描述（由申请人提供）：A-D-磷酸化酶超家族中的酶在碳水化合物代谢和其他生物合成途径中起关键作用。这些蛋白质在自然界中的分布证明了这些蛋白质的生物学重要性：在从细菌到人类的所有生物体中似乎至少有一个家庭成员。在此提案中，我们将表征该酶家族中一种蛋白质的新颖方面，磷酸胰蛋白酶/磷酸葡萄糖核糖果酶（PMM/PGM）来自铜绿假单胞菌。该细菌是一种机会主义的人类病原体，经常感染囊性纤维化患者，烧伤受害者和癌症患者。 PMM/PGM参与了多种细菌外产物的生物合成，包括脂多糖，藻酸盐和鼠李糖脂，这与生物膜形成和铜绿假单胞菌感染的毒力增加有关。由于其在毒力因子的生物合成途径中的关键作用，PMM/PGM是抑制剂设计的靶标。先前的工作表明，该酶的构象变化在其多步反应的几个关键点需要，这需要两次连续的磷酸转移反应，并在反应中间体的中间重新定位。最近，其他实验室显示了催化中新型酶动力学范式的证据：内在波动似乎逐渐催化。我们将在过程酶PMM/PGM中测试此范例。我们将使用NMR松弛分散体来表征在催化相关时间表上发生的无基质（APO）PMM/PGM的动力学。此外，我们将在其可逆催化反应期间对其动力学进行研究。我们试图确定蛋白质构象变化，毫秒内发生的固有波动以及该A-D-磷申酶酶超级家族的功能之间的关系。这将有助于开发新型抑制剂的长期目标，这些抑制剂可以在其多步反应中的不同点起作用。该提案中的工作将启动从细菌病原体铜绿假单胞菌的键酶（PMM/PGM）的动力学表征。铜绿假单胞菌感染是囊性纤维化患者死亡率的主要原因，这种生物也是医院获得感染的主要原因。 PMM/PGM的动力学研究将补充现有的结构和机械表征，其目的是设计具有效用的特定抑制剂，以治疗铜绿假单胞菌感染。