Chemistry, Structure and Biology of Thiol Transferases

硫醇转移酶的化学、结构和生物学

• 批准号: 8439114
• 负责人: RICHARD N ARMSTRONG
• 金额: $ 33.56万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8439114
• 关键词: Address; Amides; Anabolism; Antibiotics; Arachidonic Acids; Bacillus anthracis; Bacillus cereus; Binding; Biodiversity; Biology; Catabolic Process; Cells; Chemicals; Chemistry; Dependence; Deuterium; Endoplasmic Reticulum; Environment; Enzymes; Fever; Fosfomycin; Genes; Glutathione; Glutathione Metabolism Pathway; Glutathione S-Transferase; Goals; Health; Human; Hydrogen; Inflammation; Institutes; Integral Membrane Protein; Ions; Leukotrienes; Lipoxygenase; Mammals; Mass Spectrum Analysis; Membrane; Membrane Proteins; Microbe; Oxidation-Reduction; Pain; Pain management; Pathway interactions; Positioning Attribute; Prostaglandins; Proteins; Regulation; Research; Resistance; Role; Source; Staphylococcus aureus; Structure; Sulfhydryl Compounds; Transferase; Vertebral column; allergic response; chemical kinetics; design; divalent metal; inhibitor/antagonist; insight; member; microbial; microorganism; prostaglandin E synthase-1

DESCRIPTION (provided by applicant): Thiols, thiol-transferases and related proteins are essential to virtually all aspects of biology. In mammals and microbes thiols maintain redox status, protect cells against toxic electrophiles and participate in crucial biosynthetic, metaboli and catabolic processes. The general objectives of this renewal application address specific problems in the understanding of the involvement of thiol transferases and related proteins in mammalian and microbial biology. The specific aims are (i) to advance our understanding of the mechanisms and structures of three MAPEG proteins (Membrane Associated Proteins in Eicosinoid and Glutathione metabolism) and (ii) to provide previously inaccessible scientific insight into the chemistry and biology of bacillithiol (BSH); a newly discovered thiol found in Gram-positive microorganisms. Members of the MAPEG superfamily are involved in several aspects of the regulation of pain, fever, inflammation and allergic response in humans. The first specific aim focuses on crucial issues regarding three different MAPEG proteins, microsomal prostaglandin E synthase 1 (MPEGS1), 5-lipoxigenase activating protein (FLAP) and microsomal glutathione transferase 1 (MGST1). All of these proteins are integral membrane proteins. Specific Aim 1 relies heavily on hydrogen/deuterium exchange mass spectrometry (H/DEX MS) to probe the structural aspects of these proteins as they interact with substrates, inhibitors and protein partners. Specific Aim 1a will ascertain if it is possible to perform H/DEX MS on an integral membrane protein MGST1 in its native membrane environment, the endoplasmic reticulum. Specific Aim 1b will address the potential inhibition of MPGES1 by nitrosylation of C59 and the chemical mechanism of MPGES1, about which virtually nothing is known. Specific Aim 1c is will elucidate the structural details of the interaction of FLAP with arachidonic acid and its interaction with 5-lipoxigenase (5-LOX) in the initiation of the lipoxygenase pathway for the synthesis of leukotrienes. Specific aim 2 is directed at understanding the role of bacillithiol (BSH) in the resistance of Gram-positive pathogenic microorganisms to the antibiotic fosfomycin conferred by the enzyme FosB. This aim is leveraged by the fact that we have, in the last project period with help of the Vanderbilt Institut of Chemical Biology Synthesis Core, synthesized 1.3 grams bacillithiol and have obtained the genes, expressed and purified the FosB enzymes from several sources including Bacillus cereus, Bacillus anthracis and Staphylococcus aureus which puts us in an excellent position to accomplish this aim. Specific Aim 2a is to determine the crystal structure of one or more FosB proteins with fosfomycin, BSH or product bound. Specific Aim 2b will elucidate the chemistry, kinetics, and divalent metal ion-dependence of the FosB enzymes with both BSH and the alternative substrate L-Cys.

描述（由申请人提供）：硫醇，硫醇转移酶和相关蛋白质实际上对于生物学的所有方面都是必不可少的。在哺乳动物和微生物中，硫醇保持氧化还原状态，保护细胞免受有毒电力的影响，并参与至关重要的生物合成，代谢物和分解代谢过程。该更新应用程序的一般目标在理解硫醇转移酶和相关蛋白质中的哺乳动物和微生物生物学的参与时解决了特定问题。具体目的是（i）提高我们对三种MAPEG蛋白（膜相关的蛋白质和谷胱甘肽代谢中的膜相关蛋白）和（ii）的理解，以提供先前无法访问的科学洞察力，以了解芽孢杆菌的化学和生物学（BSH）；在革兰氏阳性微生物中发现的新发现的硫醇。 Mapeg超家族的成员参与了人类疼痛，发烧，炎症和过敏反应的多个方面。第一个特定目的侧重于有关三种不同的Mapeg蛋白，微粒体前列腺素E合酶1（MPEGS1），5-脂氧素激活蛋白（瓣）和微粒体谷胱甘肽转移酶1（MGST1）的关键问题。所有这些蛋白质都是整体膜蛋白。具体目标1在很大程度上依赖于氢/氘交换质谱法（H/DEX MS）来探测这些蛋白质与底物，抑制剂和蛋白质伴侣相互作用时的结构方面。具体的目标1a将确定是否可以在其天然膜环境（内质网）中对整体膜蛋白MGST1进行H/DEX MS。特定的目标1B将通过硝基化C59和MPGES1的化学机制来解决MPGES1的潜在抑制，几乎什么都不知道。具体的目标1c是将阐明皮瓣与花生四烯酸相互作用的结构细节，以及与5-脂氧酶（5-lox）相互作用在脂氧合酶合成白细胞的途径中。具体目标2是针对了解杆菌醇（BSH）在革兰氏阳性致病性微生物对酶FOSB赋予的抗生素Fosfymycin抗性中的作用。 This aim is leveraged by the fact that we have, in the last project period with help of the Vanderbilt Institut of Chemical Biology Synthesis Core, synthesized 1.3 grams bacillithiol and have obtained the genes, expressed and purified the FosB enzymes from several sources including Bacillus cereus, Bacillus anthracis and Staphylococcus aureus which puts us in an excellent position to accomplish this aim.具体目标2a是确定具有Fosfymoncin，bSH或产物结合的一个或多个FOSB蛋白的晶体结构。特定的目标2b将阐明具有BSH和替代底物L-CYS的FOSB酶的化学，动力学和二价金属离子依赖性。