Novel Catalases and their Products

新型过氧化氢酶及其产品

• 批准号: 8241151
• 负责人: ALAN R. BRASH
• 金额: $ 32.82万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8241151
• 关键词: Active Sites; Address; Aerobic; Amino Acids; Anabaena; Anabolism; Area; Atherosclerosis; Binding; Biochemistry; Biology; Carbon; Catalysis; Catalytic Domain; Characteristics; Chemistry; Cyanobacterium; Cyclization; Cytochrome P450; Cytochromes; DNA Sequence Rearrangement; Diabetes Mellitus; Dioxygenases; Disease; Enzyme Interaction; Enzymes; Epoxy Compounds; Exhibits; Family member; Fatty Acids; Felis catus; Fusarium; Grant; Heme; Hemeproteins; Hydrogen Peroxide; Hydrolysis; Hydroxylation; Inflammation; Knowledge; Life; Ligands; Light; Lipid Peroxides; Malignant Neoplasms; Metabolic; Metabolism; Mixed Function Oxygenases; Nature; Nomenclature; Nostoc; Organism; Oxidants; Oxidative Stress; Oxides; Oxygen; Peroxides; Property; Protein Family; Proteins; Pseudomonas fluorescens; Reaction; Relative (related person); Role; Sentinel; Sequence Homology; Site-Directed Mutagenesis; Specificity; Structure; Synthesis Chemistry; System; Thinking; Tyrosine; Work; allene oxide synthase; catalase; cyclooxygenase 2; cyclopentenone; enzyme activity; heme a; improved; insight; interest; novel; polypeptide; propadiene; public health relevance

DESCRIPTION (provided by applicant): The long-term objective of this application is to elucidate the roles of a novel group of proteins related in sequence and structure to catalase and to define the biochemistry and chemistry of their reactions and novel products. Catalase is renowned for its efficient reaction with hydrogen peroxide and its key role in the oxidative defense of all aerobic organisms. The catalase-related relatives studied here are smaller proteins with reaction specificity directly against fatty acid hydroperoxides. The prototypical enzyme of the proposal is an allene oxide synthase, an enzyme that catalyzes a cytochrome P450-type of reaction yet which exhibits distinct sequence homology to catalase. In Aim 1, the ability to transform the catalase-related AOS and the P450 type of AOS to monooxygenases will be examined using surrogate oxygen donors to activate the heme, with stopped flow spectral recording to detect short-lived intermediates as well as detailed product analysis to define the substrate-enzyme interaction. We also propose to change the AOS activity of these enzymes by site-direct mutagenesis of active site amino acids, a hypothesis supported by the array of products of CYP74 relatives of the P450 AOS. Aim 2 will focus on analysis of the catalytic activities of other novel enzyme candidates that have similar sequence characteristics defined as retention of the heme-binding features of a catalase within an unusually short polypeptide for catalases of only ~40kD. Aim 3 will analyze the mechanism of biosynthesis of a unique bicyclobutane fatty acid made by a catalase-related enzyme from the cyanobacterium Anabaena, and also characterize the structures of its hydrolysis and rearrangement products. This has implications regarding the potential synthesis of bicyclobutanes in other areas of biology. We will also address structural issues pertinent to the cyclization of natural allene oxide diastereomers, which is of fundamental interest in understanding the nature of allene oxide metabolism and the chemistry of cyclopentenone synthesis. The results of this study will provide new insights and a new way of thinking about the enzymatic capabilities of a long-recognized protein family with established roles as a sentinel at the forefront of oxidative defense. PUBLIC HEALTH RELEVANCE: Statement Oxidative stress is a key factor underlying the progression of many diseases ranging from atherosclerosis to diabetes, inflammation, and cancer. By uncovering the details of how different enzymes deal with oxidants and how they relate or differ from each other this project will improve our understanding of oxidative stress, which will ultimately help control it.

描述（由申请人提供）：本申请的长期目标是阐明与过氧化氢酶相关的序列和结构相关的一组新蛋白质的作用，并定义其反应和新产物的生物化学和化学。过氧化氢酶因其与过氧化氢的有效反应及其在所有需氧生物的氧化防御中的关键作用而闻名。这里研究的过氧化氢酶相关亲属是较小的蛋白质，具有直接针对脂肪酸氢过氧化物的反应特异性。该提案的原型酶是丙二烯氧化物合酶，这种酶催化细胞色素 P450 型反应，但与过氧化氢酶表现出独特的序列同源性。在目标 1 中，将使用替代氧供体激活血红素，检查将过氧化氢酶相关的 AOS 和 P450 型 AOS 转化为单加氧酶的能力，并通过停流光谱记录来检测短寿命中间体以及详细的产物分析来定义底物-酶相互作用。我们还建议通过活性位点氨基酸的定点诱变来改变这些酶的 AOS 活性，这一假设得到了 P450 AOS 的 CYP74 亲戚的一系列产物的支持。目标 2 将重点分析其他新型候选酶的催化活性，这些候选酶具有相似的序列特征，定义为在仅约 40kD 的过氧化氢酶的异常短的多肽内保留过氧化氢酶的血红素结合特征。目标 3 将分析蓝藻鱼腥藻中过氧化氢酶相关酶生物合成独特双环丁烷脂肪酸的机制，并表征其水解和重排产物的结构。这对于生物学其他领域中双环丁烷的潜在合成具有影响。我们还将解决与天然环氧丙烷非对映体环化相关的结构问题，这对于理解环氧丙烷代谢的性质和环戊烯酮合成的化学具有根本意义。这项研究的结果将为长期认识的蛋白质家族的酶促能力提供新的见解和新的思考方式，该蛋白质家族已确立了氧化防御前沿哨兵的作用。 公共健康相关性：声明氧化应激是许多疾病（从动脉粥样硬化到糖尿病、炎症和癌症）进展的关键因素。通过揭示不同酶如何处理氧化剂以及它们之间如何关联或不同的细节，该项目将增进我们对氧化应激的理解，这最终将有助于控制氧化应激。