Glutathione Transferases-Defining Structure & Function

描述(申请人提供)：生物化学的主要挑战之一是理解生物体的功能基因组学。这是一个令人震惊的问题，当人们考虑到一个事实，即大约40%的基因在最好的了解的生物之一，大肠杆菌，没有实验定义。这种微生物在出生后不久就在人体肠道定居，与其近亲如沙门氏菌、志贺氏菌和耶尔西氏菌一起，是一种病原体，是人类疾病的重要来源。谷胱甘肽(GSH)是大多数好氧生物体中主要的氧化还原活性硫醇，在代谢、分解代谢和氧化还原化学中起着重要的作用。谷胱甘肽转移酶是通过将谷胱甘肽加到亲电受体上而参与这种化学作用的酶。大肠杆菌基因组含有编码9种谷胱甘肽(GSH)转移酶同系物的基因。令人惊讶的是，只有一个基因具有相当明确的功能，它不编码GSH转移酶，而是编码严格的饥饿蛋白A，SSPA，一种转录因子。在好氧条件下，GSH是大肠杆菌中的主要硫醇。然而，在厌氧条件下，谷胱甘肽亚精胺合成酶/酰胺酶(GSS)在很大程度上转化为谷胱甘肽亚精胺(GspSH)。该项目的长期目标是了解大肠杆菌中GSH/GspSH的动态平衡，以及染色体编码的GSH转移酶同系物在大肠杆菌中的生物学活性和作用。10个相互关联的蛋白质是本次研究的对象。它们包括八个典型的GSH转移酶同源物，YliJ，YncG，GST，YfcF，YfcG，YghU，SSPA，和YbF，以及GSS，以及膜结合的GSH转移酶YecN。研究计划包括三个具体目标。第一个目的是了解大肠杆菌中GSH/GspSH动态平衡的机制和调控。第二个目的是确定八种典型的谷胱甘肽转移酶同源物在大肠杆菌中的生物学功能。第三个目的是确定单个膜结合的GSH转移酶同系物的生物学作用。这些目标将通过多学科方法实现，其中包括：(1)基因组背景分析；(2)对基因敲除的表型反应；(3)基因表达对生长条件和压力的反应；(4)寻找蛋白质伙伴；(5)确定蛋白质的X射线晶体结构；(6)蛋白质的功能和动力学特征。与公共卫生相关：大肠杆菌是肠杆菌科家族的成员，包括几种肠道病原体，如沙门氏菌、志贺氏菌和耶尔森氏菌。这种细菌通常在出生后不久就在人体肠道定居，在那里它仍然是主要的兼性微生物。了解大肠杆菌的生物化学是了解其发病机制的基础。

DESCRIPTION (provided by applicant): One of the major challenges in biochemistry is to understand the functional genomics of organisms. It is a staggering problem when one considers the fact that about 40% of the genes in of one of the best-understood organisms, Escherichia coli, are not experimentally defined. The organism colonizes the human intestine shortly after birth and is, along with its close relatives such as Salmonella, Shigella and Yersinia, a pathogen that is a significant source of human disease. Glutathione (GSH) is the predominant redox active thiol in most aerobic organisms where it plays a fundamental role in metabolic, catabolic and redox chemistry. GSH transferases are enzymes that participate in this chemistry by adding GSH to electrophilic acceptors. The E. coli genome harbors genes encoding nine glutathione (GSH) transferase homologues. Amazingly, only one gene has a reasonably well-defined function and it does NOT encode a GSH transferase but rather stringent starvation protein A, SspA, a transcription factor. Under aerobic conditions, GSH is the predominate thiol in E. coli. However it is largely converted to glutathionylspermidine (GspSH) under anaerobic conditions by glutathionylspermidine synthetase/amidase (GSS). The broad, long-term objectives of this project are to understand the GSH/GspSH homeostasis in E. coli and the biological activities and roles of chromosomally encoded GSH transferase homologues in E. coli. Ten interrelated proteins are the subject of this investigation. They include the eight canonical GSH transferase homologues, YliJ, YncG, Gst, YfcF, YfcG, YghU, SspA, and YibF as well as GSS, and the membrane-bound GSH transferase, YecN. The research plan includes three specific aims. The first aim is to understand the mechanism and regulation of GSH/GspSH homeostasis in E. coli. The second aim is to define the biological functions of the eight canonical GSH transferase homologues in E. coli. The third aim is to determine the biological role of the single membrane-bound GSH transferase homologue. These aims will be achieved by a multidisciplinary approach that includes: (i) analysis of genome context; (ii) phenotypic responses to gene knockouts; (iii) response of gene expression to growth conditions and stress; (iv) a search for protein partners; (v) determination of X-ray crystal structures of the proteins; (vi) functional and kinetic characterizations of the proteins. PUBLIC HEALTH RELEVANCE: Escherichia coli is a member of the Enterobacteriaceae family that includes several intestinal pathogens such as Salmonella, Shigella and Yersinia. The bacterium typically colonizes the human intestine shortly after birth where it remains the predominant facultative microorganism. Understanding the biochemistry of E. coli is fundamental to understanding its pathogenesis.