Novel Catalases and their Products

新型过氧化氢酶及其产品

• 批准号: 7886436
• 负责人: ALAN R. BRASH
• 金额: $ 32.94万
• 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/7886436
• 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的序列和结构相关的新型蛋白质的作用，并定义其反应和新产品的生物化学和化学。过氧化氢酶以其与过氧化氢的有效反应及其在所有有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亲属是较小的蛋白质，其反应特异性直接针对脂肪酸氢过氧化物。该提案的原型酶是一种艾琳氧化物合酶，一种酶，催化了一种反应的细胞色素P450型，但与过酶表现出不同的序列同源性。在AIM 1中，将使用替代氧供体转化与过氧化氢酶相关的AOS和P450类型的AOS变为单加氧酶，以激活血红素，并使用停止的流谱记录来检测短寿命的中间体以及详细的产品分析，以定义substrate-enzyme-Enzyme的相互作用。我们还建议通过活性位点氨基酸的位置指导诱变来改变这些酶的AOS活性，这是由P450 AOS的CYP74亲属阵列支持的假设。 AIM 2将集中于对其他新型酶的催化活性的分析，这些新酶的序列特征的定义为相似的序列特征，该序列特征的保留量仅在〜40KD的过氧化氢酶的异常短多肽中保留了血红素结合特征。 AIM 3将分析由蓝藻与蓝藻相关的酶制成的独特的双环戊烷脂肪酸的生物合成机理，并表征了其水解和重排产物的结构。这对生物学其他领域的骑自行车型的潜在合成具有含义。我们还将解决与自然氧化物非对映异构体的环化有关的结构性问题，这对于理解艾琳氧化物代谢的性质和环戊酮合成的化学性质具有根本兴趣。这项研究的结果将提供新的见解和一种新的思考方式，以了解长期认可的蛋白质家族的酶促能力，该家族在氧化防御的最前哨中既定角色。 公共卫生相关性：陈述氧化应激是从动脉粥样硬化到糖尿病，炎症和癌症的许多疾病进展的关键因素。通过揭示不同酶如何处理氧化剂以及它们如何相互联系或彼此之间的细节，该项目将改善我们对氧化应激的理解，这最终将有助于控制它。