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: 7293830
• 负责人: LESA J BEAMER
• 金额: $ 7.48万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7293830
• 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; Peptidylprolyl Isomerase; Phosphoglucomutase; Phosphomannomutase; Play; Protein Dynamics; Proteins; Pseudomonas aeruginosa; Rate; 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）：α-D-磷酸己糖变位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发挥关键作用。这些蛋白质的生物学重要性通过它们在自然界中的分布得到了证明：从细菌到人类的所有生物体中似乎都发现了至少一种家族成员。在本提案中，我们将表征该酶家族中一种蛋白质的新特征，即来自铜绿假单胞菌的磷酸甘露糖变位酶/磷酸葡萄糖变位酶（PMM/PGM）。这种细菌是一种机会性人类病原体，经常感染囊性纤维化患者、烧伤患者和癌症患者。 PMM/PGM 参与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，它们与铜绿假单胞菌感染中生物膜的形成和毒力增加有关。由于其在毒力因子生物合成途径中的关键作用，PMM/PGM 是抑制剂设计的目标。先前的工作表明，该酶的多步反应中的几个关键点需要构象变化，这需要两个连续的磷酰基转移反应以及反应中间体的重新定向。最近，其他实验室已经证明了催化中酶动力学的新范式：内在波动似乎与催化耦合。我们将在加工酶 PMM/PGM 中测试这一范例。我们将使用 NMR 弛豫色散来表征在催化相关时间尺度上发生的无底物 (apo) PMM/PGM 的动力学。此外，我们将开始研究其可逆催化反应过程中的动力学。我们试图确定蛋白质构象变化、毫秒内发生的内在波动以及α-D-磷酸己糖变位酶超家族成员的功能之间的关系。这将有助于实现开发新型抑制剂的长期目标，这些抑制剂可以在多步反应的不同点发挥作用。该提案中的工作将启动对细菌病原体铜绿假单胞菌的关键酶 (PMM/PGM) 动力学的表征。铜绿假单胞菌感染是囊性纤维化患者死亡的主要原因，也是医院获得性感染的主要原因。 PMM/PGM 的动力学研究将补充现有的结构和机制表征，目标是设计可用于治疗铜绿假单胞菌感染的特定抑制剂。