McVey Alyssa C, Medarametla Prasanthi, Chee Xavier, Bartlett Sean, Poso Antti, Spring David R, Rahman Taufiq, Welch Martin
Department of Biochemistry, University of Cambridge , Cambridge CB2 1QW, U.K.
School of Pharmacy, University of Eastern Finland , 70211 Kuopio, Finland.
Biochemistry. 2017 Oct 17;56(41):5539-5549. doi: 10.1021/acs.biochem.7b00852. Epub 2017 Oct 6.
Pseudomonas aeruginosa is an opportunistic human pathogen recognized as a critical threat by the World Health Organization because of the dwindling number of effective therapies available to treat infections. Over the past decade, it has become apparent that the glyoxylate shunt plays a vital role in sustaining P. aeruginosa during infection scenarios. The glyoxylate shunt comprises two enzymes: isocitrate lyase and malate synthase isoform G. Inactivation of these enzymes has been reported to abolish the ability of P. aeruginosa to establish infection in a mammalian model system, yet we still lack the structural information to support drug design efforts. In this work, we describe the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. The enzyme is a monomer composed of four domains and is highly conserved with homologues found in other clinically relevant microorganisms. It is also dependent on Mg for catalysis. Metal ion binding led to a change in the intrinsic fluorescence of the protein, allowing us to quantitate its affinity for Mg. We also identified putative drug binding sites in malate synthase G using computational analysis and, because of the high resolution of the experimental data, were further able to characterize its hydration properties. Our data reveal two promising binding pockets in malate synthase G that may be exploited for drug design.
铜绿假单胞菌是一种机会性人类病原体，由于可用于治疗感染的有效疗法数量不断减少，被世界卫生组织认定为重大威胁。在过去十年中，已明显看出乙醛酸循环途径在感染情况下维持铜绿假单胞菌的生存方面起着至关重要的作用。乙醛酸循环途径由两种酶组成：异柠檬酸裂解酶和苹果酸合酶同工型G。据报道，这些酶的失活会消除铜绿假单胞菌在哺乳动物模型系统中建立感染的能力，但我们仍然缺乏支持药物设计工作的结构信息。在这项工作中，我们描述了铜绿假单胞菌苹果酸合酶G无配体形式的首个X射线晶体结构，分辨率为1.62 Å。该酶是由四个结构域组成的单体，与其他临床相关微生物中的同源物高度保守。它的催化作用也依赖于镁。金属离子结合导致蛋白质内在荧光发生变化，使我们能够定量其对镁的亲和力。我们还通过计算分析在苹果酸合酶G中确定了假定的药物结合位点，并且由于实验数据的高分辨率，进一步能够表征其水合性质。我们的数据揭示了苹果酸合酶G中两个有前景 的结合口袋，可用于药物设计。
