Mechanistic Studies of TfdA, an Alpha-Ketoglutarate- Dependent Dioxygenase

TfdA（一种α-酮戊二酸依赖性双加氧酶）的机制研究

• 批准号: 9603520
• 负责人: Robert Hausinger
• 金额: $ 27万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-15 至 2000-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9603520&HistoricalAwards=false
• 关键词: Mechanistic; Studies; TfdA; Alpha; Ketoglutarate

Hausinger 96-03520 Hausinger Part 1. Technical This study uses 2,4-dichlorophenoxyacetic acid (2,4-D)/alpha-keto glutarate dependent dioxygenase (tfdA) from Alcaligenes eutrophus as a model protein to examine the metallocenter properties and enzyme mechanisms in catalysis. This enzymes catalyzes the first step in biodegradation of the broadleaf herbicide 2,4-D. It couples the hydroxylation of 2,4-D, forming a compound that spontaneously decomposes to yield glyoxylate and 2,4-dichlorophenol, with the conversion of alpha-KG to CO2 and succinate in a ferrous ion-dependent reaction. TfdA is especially well suited to serve as a paradigm of this enzyme class because it is relatively small, highly soluble, end readily available in large quantities by using recombinant cell extracts and a simple two-step purification protocol. Furthermore, the tfdA gene is easily manipulated by genetic and recombinant methods. The objectives and approaches are: (1) characterize the metallocenter in TfdA bond to Fe(Il) or substituted metals by spectroscopic methods such as , X-ray absorption, and electron paramagnetic resonance ; (2) Identify the residues that participate as metallocenter or in substrate or cofactor binding and enzymatic catalysis by site-directed mutagenesis; (3) define the enzyme mechanism by examining the reactivity of TfdA towards alternate substrates, inhibitors, and inactivators; and (4) initiate studies on a related protein from E. coli, TauD, a sulfonate hydroxylase that degrades sulfonates to form sulfite, the cellular sulfur source. Results from these studies, designed to further the understanding of the novel biodegradative chemistry of TfdA, and other enzymes, may be useful to biotechnological and bioremediative applications such as the selective hydroxylation or decomposition of other compounds. Part 2. Non-technical This project seeks to characterize the enzymatic mechanism of TfdA, an enzyme that catalyzes the first step in the decomposition of the broadleaf herbicide 2,4D (2,4-d ichlorophenoxyacetic acid), from Alcaligenes eutrophus. TfdA is a representative of an important, yet poorly understood, class of ferrous ion-dependent enzymes that couple the oxygen-requiring decomposition of alpha-ketoglutarate to the hydroxylation of a substrate. Changes occurring at the metallocenter will be examined by spectroscopy, the roles of selected amino acid residues studied by molecular biological methods and the enzyme mechanism deduced by analyzing its reactivity towards substrates, inhibitors, and inactivators. In addition, the properties of this enzyme will be compared to those proteins related in sequence. Results from these studies should advance the understanding of the novel reaction mechanisms of enzymes useful in biotechnology and biodegradation. 80 Fane The proper assembly of viral proteins and nucleic acids into a biologically active virion involves numerous and diverse macromolecular interactions. The main objectives are to elucidate these critical interactions and to define the structural domains of the responsible macromolecules. A combination of genetic, biochemical and structural approaches (X-ray crystallography) will be employed to accomplish the object. Like molecular chaperones, scaffolding proteins direct other proteins in achieving their proper three-dimensional conformations. Within the context of this analogy, the atomic structure of a procapsid offers a depiction of a chaperone-like protein complexed with its substrate. Prior results suggest that the Microviridae internal scaffolding proteins, gpB, share many properties with molecular chaperones. Prior results also indicate that the internal scaffolding proteins either possess inherent flexibility or interact with their substrates in nonspecific manners, perhaps via interfaces. Determining the atomic structures of hybrid procapsids, containing foreign scaffolding proteins, will directly address this questio n. The present (X174 procapsid structure does not contain the internal scaffolding protein which is lost during purification. During the first year of support, alternate genetic and purification strategies will be explored to stabilize this protein. The current protocols will still be employed to gather additional data to refine the external scaffolding protein structure. Also within the first year, the genetic and purification strategies needed to generate alpha procapsids will be developed. Continuation of the structural work, done in collaboration with Dr. M. G. Rossmann, will proceed throughout the support period. The recently solved atomic structure of the external scaffolding protein, gpD, suggests that it also shares many features with molecular chaperones. Thus gpD, like a molecular chaperone, can bind to other proteins in many different ways. While some domains make contact with the coat protein in varied manners, one of these domains may determine the protein's substrate specificity for a particular viral coat protein. the plasmid-based cross complementation system has been extended to this gene. Unlike the internal scaffolding proteins which exhibit a great deal of divergence in primary structure, the external scaffolding proteins share 75% sequence identity. The (X174 protein, however, is unable to productively direct the assembly of other Microviridae virions. A comparison of the primary structure reveals that the divergent residues are localized to the NH2 -termini of the proteins which forms a large (-helix in the (X174 atomic structure. These observations suggest a model in which the scaffolding's specificity for a particular viral coat protein resides in this region. This hypothesis will be tested with chimeric polypeptides. During the first year of support, restriction sites needed to construct chimeric external scaffolding proteins will be introduced. The (X174 gene will be recloned and clones of other Microviridae D genes will be generated. Characterization of the chimeric genes and gene products will commence in the second year. The results of these analyses may also provide insights into the design of recombinant proteins. All viruses must assemble themselves by means of multiple protein interactions. viral assembly is often dependent on proteins known as scaffolding proteins. Analogous to scaffoldings used in the construction of buildings, scaffolding proteins are found in virus assembly intermediates but not in the mature viruses. Two different scaffolding proteins, external and internal, are required for the assembly of the Microviridae family of viruses. With These viruses we are able to purify viral intermediates which still include the external scaffolding protein. By examining the atomic structure of the external scaffolding protein and performing genetic analyses with both the internal and external proteins, we have determined that different regions of the scaffolding proteins may have specific and identifiable functions. Refining the atomic structure of these proteins and testing our hypotheses regarding their various functions by constructing hybrid Microviridae scaffolding proteins are the main objectives of the proposed work. The results of these analyses will provide further insights into virus assembly and the design of recombinant proteins.

Hausinger 96-03520 Hausinger Part 1。本研究以富营养化海藻中的2,4-二氯苯氧乙酸(2,4- d)/ α -酮戊二酸盐依赖双加氧酶（tfdA）为模型蛋白，研究了金属中心性质和酶催化机制。这种酶催化了阔叶除草剂2,4- d生物降解的第一步。它结合了2,4-d的羟基化，形成一种自发分解生成乙醛酸酯和2,4-二氯苯酚的化合物，并在依赖铁离子的反应中将- kg转化为CO2和琥珀酸盐。TfdA特别适合作为这类酶的典范，因为它相对较小，高可溶性，易于通过重组细胞提取物和简单的两步纯化方案大量获得。此外，tfdA基因易于通过遗传和重组方法进行操作。目的和方法是：(1)通过x射线吸收和电子顺磁共振等光谱方法表征TfdA与Fe（Il）或取代金属键中的金属中心；(2)通过位点导向诱变确定作为金属中心或参与底物或辅因子结合和酶催化的残基；(3)通过检测TfdA对替代底物、抑制剂和灭活剂的反应性来确定酶的机制；(4)启动大肠杆菌相关蛋白TauD的研究，TauD是一种磺酸羟化酶，可降解磺酸盐形成亚硫酸盐，亚硫酸盐是细胞硫源。这些研究的结果旨在进一步了解TfdA和其他酶的新型生物降解化学，可能对生物技术和生物修复应用（如选择性羟基化或其他化合物的分解）有用。第2部分。该项目旨在表征TfdA的酶促机制，TfdA是一种酶，它催化富营养盐中阔叶除草剂2,4d （2,4-d氯苯氧乙酸）分解的第一步。TfdA是一类重要但鲜为人知的亚铁离子依赖性酶的代表，它将α -酮戊二酸的需要氧分解与底物的羟基化结合在一起。金属中心发生的变化将通过光谱学进行检测，通过分子生物学方法研究选定的氨基酸残基的作用，并通过分析其对底物、抑制剂和灭活剂的反应性推断酶的机制。此外，将该酶的性质与序列相关的蛋白质进行比较。这些研究结果将促进对生物技术和生物降解中有用的酶的新反应机制的理解。80 .将病毒蛋白和核酸正确组装成具有生物活性的病毒粒子涉及许多不同的大分子相互作用。主要目的是阐明这些关键的相互作用，并定义负责大分子的结构域。遗传学、生物化学和结构方法（x射线晶体学）的结合将被用来完成这个目标。像分子伴侣一样，支架蛋白指导其他蛋白质获得适当的三维构象。在这种类比的背景下，原衣壳的原子结构提供了一种与底物复合的伴侣样蛋白质的描述。先前的研究结果表明，微病毒科内部支架蛋白（gpB）与分子伴侣蛋白具有许多相同的特性。先前的结果还表明，内部支架蛋白要么具有固有的灵活性，要么以非特异性的方式与底物相互作用，可能通过界面。确定含有外源支架蛋白的杂化原衣壳的原子结构将直接解决这一问题。目前的X174原衣壳结构不含纯化过程中丢失的内部支架蛋白。在支持的第一年，将探索替代的遗传和纯化策略来稳定这种蛋白质。目前的方案仍将用于收集额外的数据，以完善外部支架蛋白结构。同样在第一年，产生α原囊体所需的遗传和纯化策略将被开发出来。与m·g·罗斯曼博士合作进行的结构工作将在整个支助期间继续进行。最近发现的外部支架蛋白gpD的原子结构表明，它与分子伴侣蛋白有许多共同的特征。因此，像分子伴侣一样，gpD可以以许多不同的方式与其他蛋白质结合。虽然一些结构域以不同的方式与外壳蛋白接触，但其中一个结构域可能决定蛋白质对特定病毒外壳蛋白的底物特异性。基于质粒的交叉互补系统已扩展到该基因。与内部支架蛋白在一级结构上存在很大差异不同，外部支架蛋白具有75%的序列同一性。然而，（X174）蛋白不能有效地指导其他微病毒科病毒粒子的组装。初级结构的比较表明，不同的残基定位于蛋白质的NH2末端，在X174原子结构中形成一个大的螺旋。这些观察结果提示了一个模型，其中支架对特定病毒外壳蛋白的特异性驻留在该区域。这一假设将用嵌合多肽进行验证。在第一年的支持中，将引入构建嵌合外部支架蛋白所需的限制性位点。（X174）基因将被重新克隆，其他微病毒科D基因的克隆将被生成。嵌合基因和基因产物的表征将在第二年开始。这些分析的结果也可能为重组蛋白的设计提供见解。所有的病毒都必须通过多种蛋白质的相互作用来组装自己。病毒组装通常依赖于被称为支架蛋白的蛋白质。类似于建筑中使用的支架，支架蛋白存在于病毒组装中间体中，但不存在于成熟病毒中。两种不同的支架蛋白，外部和内部，是组装微病毒科病毒所需的。有了这些病毒，我们就能够提纯病毒中间产物，其中仍然包括外部支架蛋白。通过检查外部支架蛋白的原子结构，并对内部和外部蛋白进行遗传分析，我们确定了支架蛋白的不同区域可能具有特定的可识别的功能。完善这些蛋白质的原子结构，并通过构建混合微病毒科支架蛋白来测试我们关于其各种功能的假设是本研究的主要目标。这些分析的结果将为病毒组装和重组蛋白的设计提供进一步的见解。