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(申请人提供)：本申请的长期目标是阐明一组在序列和结构上与过氧化氢酶相关的新蛋白质的作用，并确定其反应和新产品的生化和化学。过氧化氢酶以其与过氧化氢的高效反应和在所有需氧生物的氧化防御中的关键作用而闻名。这里研究的与过氧化氢酶相关的是一些较小的蛋白质，它们具有直接对抗脂肪酸氢过氧化物的反应专一性。该提案的原型酶是丙二烯氧化物合成酶，这是一种催化细胞色素P450类型的反应的酶，但它与过氧化氢酶具有明显的序列同源性。在目标1中，将使用替代供氧体来激活血红素，并使用停止流动光谱记录来检测短暂的中间体，以及详细的产品分析来确定底物-酶的相互作用，以检验将过氧化氢酶相关的AOS和P450类型的AOS转化为单加氧酶的能力。我们还建议通过活性部位氨基酸的定点突变来改变这些酶的AOS活性，这一假设得到了P450 AOS的CYP74相关产物阵列的支持。目的2将重点分析其他新的候选酶的催化活性，这些候选酶具有类似的序列特征，即对于~40kD的过氧化氢酶来说，在异常短的多肽中保留了过氧化氢酶的血红素结合特征。目的3分析鱼腥藻过氧化氢酶相关酶生物合成一种独特的双环丁烷脂肪酸的机理，并对其水解物和重排产物的结构进行表征。这对双环丁烷在生物学其他领域的潜在合成具有重要意义。我们还将讨论与天然环氧丙烯非对映异构体环化有关的结构问题，这对理解环氧丙烯代谢的性质和环戊酮合成的化学具有重要意义。这项研究的结果将提供新的见解和新的思维方式来研究一个被长期认可的蛋白质家族的酶功能，该家族被确立为氧化防御前沿的哨兵。 公共卫生相关性：声明氧化应激是导致从动脉粥样硬化到糖尿病、炎症和癌症等多种疾病进展的关键因素。通过揭示不同的酶如何处理氧化剂的细节，以及它们如何相互联系或不同，这个项目将提高我们对氧化应激的理解，这最终将有助于控制氧化应激。