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

描述（由申请人提供）：a- d -磷酸己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发挥关键作用。这些蛋白质在自然界中的分布证明了它们的生物学重要性：从细菌到人类的所有生物体中似乎至少有一个家族成员。在这篇文章中，我们将描述该酶家族中一个蛋白的新特性，即来自铜绿假单胞菌的磷酸甘露氨脲酶/磷酸葡萄糖糖脲酶（PMM/PGM）。这种细菌是一种机会性的人类病原体，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、海藻酸盐和鼠李糖脂，这些外产物与铜绿假单胞菌感染的生物膜形成和毒力增加有关。由于其在毒力因子的生物合成途径中起着关键作用，PMM/PGM是抑制剂设计的靶标。先前的研究表明，这种酶的构象变化在其多步反应的几个关键点上是必需的，这需要两个连续的磷酸化转移反应和中间反应的重定向。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。我们将在进程酶PMM/PGM中测试这一范式。我们将使用核磁共振弛豫色散来表征在催化相关时间尺度上发生的无底物（载子）PMM/PGM的动力学。此外，我们将开始对其可逆催化反应动力学进行研究。我们试图确定蛋白质构象变化之间的关系，在毫秒内发生的内在波动，以及a-D-磷酸己糖酶超家族成员的功能。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骤反应的不同点起作用。本研究将对铜绿假单胞菌关键酶PMM/PGM的动力学特性进行初步研究。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，目的是设计特异性抑制剂，用于治疗铜绿假单胞菌感染。

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.