Novel Catalases and their Products

描述（由申请人提供）：本申请的长期目标是阐明序列和结构与过氧化氢酶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的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，一种催化细胞色素P450型反应的酶，但与过氧化氢酶具有明显的序列同源性。在目标1中，将使用替代氧供体来激活血红素，使用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确定底物-酶相互作用，来检查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CYP 74亲属产品阵列的支持。目标2将集中于分析其他新的候选酶的催化活性，这些候选酶具有相似的序列特征，定义为过氧化氢酶的血红素结合特征保留在仅约40 kD的异常短的多肽内。目的3分析鱼腥藻过氧化氢酶合成双环丁烷脂肪酸的机制，并对其水解和重排产物的结构进行表征。这对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丙二烯氧化物非对映异构体的环化有关的结构问题，这对理解丙二烯氧化物代谢的性质和环戊烯酮合成的化学性质具有根本意义。这项研究的结果将提供新的见解和一种新的思考方式，即长期公认的蛋白质家族的酶促能力，该蛋白质家族在氧化防御的最前沿具有确定的哨兵作用。 公共卫生相关性：氧化应激是许多疾病进展的关键因素，从动脉粥样硬化到糖尿病，炎症和癌症。通过揭示不同酶如何处理氧化剂的细节以及它们如何相互关联或不同，该项目将提高我们对氧化应激的理解，最终有助于控制它。

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.