Structural Studies on Heme Containing Enzymes

含血红素酶的结构研究

• 批准号: 9405218
• 负责人: Thomas Poulos
• 金额: $ 44.5万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-15 至 1998-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9405218&HistoricalAwards=false
• 关键词: Structural; Studies; Heme; Containing; Enzymes

9405281 Poulos The overall goal of this project is to further our understanding on the mechanism of heme containing enzymes using protein crystallography as our main tool. These enzymes carry out two processes fundamental to all living systems. First, the heme group activates either O2 or H2O2 and stores the oxidizing equivalents of the oxygen or peroxide in the active site. Second, the oxidizing equivalents now stored at the heme active center are used to oxidize other molecules. These other molecules can be another redox protein or an organic molecule. Our goal is to compare several crystal structures of different heme enzymes in order to understand how these enzymes catalyze cleavage of the peroxide or O2 O-O bond. Because the heme group and its activated oxointermediates exhibit distinct spectral properties, it is possible to correlate the crystal structure information with the spectral data to obtain a deeper insight into the precise mechanism of oxygen activation and the structure of the resulting activated intermediate. The second part of the reaction involves oxidation of substrate by the enzyme. If the substrate is another protein, the problem becomes one of protein-protein recognition and long-range electron transfer through proteins. Small molecule oxidation also involves electron transfer from the substrate to the enzyme but in this case, we expect the enzyme to possess a more conventional small molecule binding pocket which specifically recognizes the substrate. Comparative crystal structure information is essential to understand the diversity of reactions and substrates used by various peroxidases and oxygenases. %%% Peroxidases and oxidases also are attracting increasing attention by the biotechnology community. These enzymes are, in some cases, able to oxidize toxic pollutants such as chlorinated biphenyls. One of the more important advances in this area was our recent structure determination of lignin peroxidases supported by NSF. Lignin perox idase is secreted by certain fungi that are able to degrade lignin, a complex aromatic polymer that provides the outer coating around cellulose in woody plants. Lignin is the second most abundant polymer on earth and presents a major problem in the paper pulp industry since lignin degradation requires rather harsh chemical treatments. There is considerable interest in developing less harsh biological processes for lignin degradation. Moreover, because of the unusual reactivity of lignin peroxidase, there is optimism that this peroxidase can be utilized for the degradation of toxic pollutants. Our structural work has provided the basis for understanding how such reactions occur. Of particular importance are recent developments in our ability to clone and overexpress heme enzymes. This opens the way for using protein engineering to alter the active sites of peroxidases and other heme enzymes in order to optimize their use for degrading molecules of interest such as toxic pollutants. The crystal structure data is essential for such an undertaking. It also should be emphasized that over the years, NSF has been the primary means of support for heme enzyme crystallography. The first two heme enzyme crystal structures solved were cytochrome c peroxidase and cytochrome P450, both of which were solved in our lab with support provided by NSF. These two structures alone have provided the basis for research in dozens of other labs. and countless spin-off projects. NSF also supported solution of the lignin peroxidase structure. Hence, it is not an understatement to claim that NSF is directly responsible for a majority of what we know today about heme enzyme structure and function and it is very likely that there will be significant and important practical applications of this work in the near future. ***

9405281 POULOS这个项目的总体目标是进一步了解使用蛋白质晶体学作为我们的主要工具的血红素机理的理解。这些酶进行了两个对所有生物系统基础的过程。首先，血红素组激活O2或H2O2，并在活性位点存储氧或过氧化物的氧化当量。其次，现在存储在血红素活性中心的氧化等效物用于氧化其他分子。这些其他分子可以是另一种氧化还原蛋白或有机分子。我们的目标是比较不同血红素酶的几种晶体结构，以了解这些酶如何催化过氧化物或O2 O-O键的裂解。由于血红素组及其活化的氧化中间体具有独特的光谱特性，因此有可能将晶体结构信息与光谱数据相关联，以更深入地了解氧气激活的精确机制以及结果激活的中间体的结构。反应的第二部分涉及酶氧化底物。如果底物是另一种蛋白质，则该问题成为蛋白质 - 蛋白质识别和通过蛋白质的远距离电子转移之一。小分子氧化也涉及从底物到酶的电子转移，但是在这种情况下，我们希望该酶具有更常规的小分子结合袋，该袋专门识别底物。比较晶体结构信息对于了解各种过氧化物酶和氧酶使用的反应和底物的多样性至关重要。 %% %%过氧化物酶和氧化酶也吸引了生物技术界的越来越多的关注。在某些情况下，这些酶能够氧化有毒污染物，例如氯化二苯基。该领域最重要的进步之一是我们最近对NSF支持的木质素过氧化物酶的结构确定。 木质素过氧ID酶由某些真菌分泌，这些真菌能够降解木质素，木质素是一种复杂的芳香族聚合物，可在木本植物的纤维素周围提供外涂层。木质素是地球上第二大的聚合物，并且在纸浆工业中提出了一个主要问题，因为木质素降解需要相当严厉的化学处理。人们对为木质素降解的较少苛刻的生物学过程产生了极大的兴趣。此外，由于木质素过氧化物酶的异常反应性，因此人们认为该过氧化物酶可以用于降解有毒污染物。我们的结构工作为理解这种反应的发生提供了基础。我们克隆和过表达血红素酶的能力的最新发展尤为重要。这为使用蛋白质工程改变过氧化物酶和其他血红素酶的活性位点开辟了道路，以优化其用于降解感兴趣的分子（例如有毒污染物）的使用。晶体结构数据对于这样的事业至关重要。还应强调的是，多年来，NSF一直是支持血红素酶晶体学的主要手段。 求解的前两个血红素酶晶体结构是细胞色素C过氧化物酶和细胞色素P450，这两种酶在我们的实验室中均得到了NSF的支持。 仅这两个结构就为其他数十个实验室的研究提供了基础。和无数的衍生项目。 NSF还支持木质素过氧化物酶结构的溶液。 因此，声称NSF直接负责我们今天对血红素酶结构和功能的大部分内容直接负责并不是一件轻描淡写的事情，而且很可能在不久的将来会有这项工作的重要而重要的实用应用。 ***