MECHANISTIC ANALYSIS OF PREPHENATE DEHYDOGENASE, SHIKIMATE DEHYDROGENASE

预酚酸脱氢酶、莽草酸脱氢酶的机理分析

基本信息

批准号：
8363550
负责人：
DINESH CHRISTENDAT
金额：
$ 1.09万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8363550
关键词：
Active Sites Alkaloids Anabolism Antibiotic Resistance Antibiotics Apicomplexa Aromatic Amino Acids Aromatic Compounds Auxins Bacteria Biochemistry Biological Biological Process Cells Commit Complex Crystallization Data Collection Decarboxylation Dehydration Development Drug Compounding Drug Efflux Enterobacteriaceae Enzymes Flavonoids Folate Funding Germination Goals Grant Growth Hydro-Lyases Ligands Metabolic Pathway Microbe National Center for Research Resources Organism Parasites Pathway interactions Pharmaceutical Preparations Phenylalanine Plants Play Pollen Prephenate Dehydrogenase Principal Investigator Protein Biosynthesis Pyruvate Quinones Reaction Repressor Proteins Research Research Infrastructure Resources Role Route Signal Transduction Source Structure Time Tyrosine United States National Institutes of Health Variant Vitamins Yeasts antimicrobial cost cyanogenic glycosides design efflux pump enzyme mechanism enzyme substrate microorganism mutant plant fungi prephenate shikimate shikimate dehydrogenase stem

Active Sites Alkaloids Anabolism Antibiotic Resistance Antibiotics Apicomplexa Aromatic Amino Acids Aromatic Compounds Auxins Bacteria Biochemistry Biological Biological Process Cells Commit Complex Crystallization Data Collection Decarboxylation Dehydration Development Drug Compounding Drug Efflux Enterobacteriaceae Enzymes Flavonoids Folate Funding Germination Goals Grant Growth Hydro-Lyases Ligands Metabolic Pathway Microbe National Center for Research Resources Organism Parasites Pathway interactions Pharmaceutical Preparations Phenylalanine Plants Play Pollen Prephenate Dehydrogenase Principal Investigator Protein Biosynthesis Pyruvate Quinones Reaction Repressor Proteins Research Research Infrastructure Resources Role Route Signal Transduction Source Structure Time Tyrosine United States National Institutes of Health Variant Vitamins Yeasts antimicrobial cost cyanogenic glycosides design efflux pump enzyme mechanism enzyme substrate microorganism mutant plant fungi prephenate shikimate shikimate dehydrogenase stem

展开

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Prephenate dehydrogenase: The first committed step in tyrosine biosynthesis is the oxidative decarboxylation of prephenate to p-hydroxylphenylpyruvate (HPP). This reaction is catalyzed by prephenate dehydrogenase in the presence of NAD+ [1]. This enzyme is of paramount importance since it channels prephenate, a branch point intermediate in tyrosine and phenylalanine biosynthesis, to tyrosine synthesis. The biosynthesis of tyrosine is of critical importance for the growth and survival of enteric bacteria, yeasts, fungi and plants. Like the other aromatic amino acids, tyrosine plays dual roles in the biochemistry of the organism, acting as both a product and a precursor. In the former case, tyrosine is required for protein synthesis, whereas, in the latter example, it is a substrate for enzymes in downstream metabolic pathways. The aromatic metabolites derived from tyrosine include quinones [2, 3], cyanogenic glycosides [4], alkaloids [5, 6], flavonoids [7], and phenolic compounds derived from the phenylpropanoid pathway [7, 8]. Since many of these compounds are involved in primary biological processes, they are essential for viability. In plants, for example, flavonoids are important for normal development as they are involved in auxin transport [9-11], pollen germination [9, 12, 13], and signaling to symbiotic microorganisms [9, 14]. We have recently determined the crystal structure of prephenate dehydrogenase however we propose that our understanding of the mechanism of this enzyme hinges on determining its crystal structure in complex with know ligands. We recently obtained crystals of prephenate dehydrogenase in co-crystallization studies using prephenate, (4-hydroxyphenyl)pyruvate and tyrosine separately. We are requesting beam time for x-ray data collection to determine the structure of prephenate dehydrogenase in complex with these ligands. Shikimate dehydrogenase: The shikimate pathway is involved in the biosynthesis of the aromatic amino acids, folates, vitamins, quinones and a variety of other aromatic compounds in bacteria, plants, fungi and apicomplexa parasites. Some of the aromatic compounds are essential for the survival of these organisms; as a result, the shikimate pathway has been an attractive target for the design of antimicrobial and herbicidal agents. This bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate followed by the reduction of dehydroshikimate to shikimate in the shikimate pathway. We have recently determined the crystal structure of DHQ-SDH. We have recently made a number of active site mutants, which we proposed are important for our understanding of the catalytic mechanism of this enzyme. We have now obtained crystals of these mutant variants and are requesting beamtime for x-ray data collection. MarR: The biological route by which an organism acquires antibiotic resistance can stem from one or more mechanisms, most of which are not well understood. One such mechanism is multiple antibiotic resistance (MAR) in which microbes reduce their intracellular concentration of antibiotics by upregulating the expression of drug efflux pumps, which actively remove drug-like compounds from the cell . We have recently determined the crystal structure of a multiple antibiotic resistance repressor protein (marR) and have conducted co-crystallization studies with a number of drug compounds. We have obtained crystals of marR in a number of co-crystallization studies and are requesting beamtime for x-ray data collection. Our goal is to determine the structure of marR in complex with the different drug compounds, which is important for our understanding of the mechanim of multiple antibiotic resistance.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。对该子项目的主要支持和子弹的主要研究者可能由其他来源（包括其他NIH来源）提供。该子项目列出的总成本可能代表了子项目使用的估计中心基础设施的估计数量，而不是NCRR赠款向子项目或副标理人员提供的直接资金。前苯甲酸苯甲酸酯脱氢酶：酪氨酸生物合成的第一个投入步骤是将前苯甲酸苯甲酸苯甲酸二苯二羧酸脱羧为P-羟基苯基丙酮酸（HPP）。在NAD+存在的情况下，该反应被前苯甲酸甲酸酯脱氢酶催化[1]。这种酶至关重要，因为它将prephenate（酪氨酸和苯丙氨酸生物合成中的一个中间分支）引导到酪氨酸合成中。酪氨酸的生物合成对于肠细菌，酵母，真菌和植物的生长和存活至关重要。像其他芳香氨基酸一样，酪氨酸在生物体的生物化学中起双重作用，既是产物又是前体。在前一种情况下，酪氨酸是蛋白质合成所必需的，而在后一个例子中，它是下游代谢途径中酶的底物。源自酪氨酸的芳族代谢产物包括奎因酮[2，3]，氰化糖苷[4]，生物碱[5，6]，类黄酮[7]和酚类化合物[7]和酚类化合物[7，8]。由于这些化合物中的许多都参与了主要的生物学过程，因此它们对于生存能力至关重要。例如，在植物中，类黄酮对正常发育很重要，因为它们参与了生长素转运[9-11]，花粉发芽[9，12，13]，以及对共生微生物的信号[9，14]。我们最近已经确定了前苯甲酸脱氢酶的晶体结构，但是我们提出，我们对这种酶的机制的理解呈现在确定其在复合物中的晶体结构与知识配体中。最近，我们在共结晶研究中使用前苯甲酸盐，（4-羟基苯基）丙酮酸和酪氨酸分别获得了前苯甲酸脱氢酶的晶体。我们要求X射线数据收集的光束时间，以确定与这些配体复合物中的前苯甲酸脱氢酶的结构。光滑的脱氢酶：Shikimate途径参与芳香氨基酸，叶酸，维生素，奎因酮以及细菌，植物，真菌和Apicomplexa寄生虫中各种其他芳香族化合物的生物合成。某些芳香化合物对于这些生物的生存至关重要。结果，光滑的途径一直是设计抗菌和除草剂剂的吸引力。这种双功能酶脱氢酶脱水酶酸性脱氢酶（DHQ-SDH）催化脱氢Qualine的脱氢脱氢，然后将脱氢丝状降低至Shikimate途径中的Shikimate。我们最近确定了DHQ-SDH的晶体结构。我们最近制作了许多活跃的位点突变体，我们提出这对于我们对这种酶的催化机制的理解很重要。现在，我们已经获得了这些突变变体的晶体，并要求X射线数据收集的Beamtime。 MARR：生物体获得抗生素耐药性的生物学途径可能源于一种或多种机制，其中大多数机制尚不清楚。一种这种机制是多种抗生素耐药性（MAR），其中微生物通过上调药物外排泵的表达来降低其细胞内抗生素的浓度，从而积极去除细胞中的药物样化合物。我们最近确定了多种抗生素抗性抑制剂蛋白（MARR）的晶体结构，并与多种药物化合物进行了共结晶研究。在许多共结晶研究中，我们已经获得了MARR的晶体，并要求X射线数据收集。我们的目标是确定MARR与不同药物化合物复杂化的结构，这对于我们对多种抗生素耐药性机械的理解非常重要。