MECHANISTIC ANALYSIS OF PREPHENATE DEHYDOGENASE, SHIKIMATE DEHYDROGENASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。对该分项目和该分项目首席调查员的主要支持可能是由其他来源提供的，包括国家卫生研究院的其他来源。列出的子项目总成本可能代表子项目使用的中心基础设施的估计数量，而不是NCRR拨款提供给子项目或子项目工作人员的直接资金。 预苯酸脱氢酶：酪氨酸生物合成的第一步是预苯酸氧化脱酸为对羟基苯基丙酮酸(HPP)。在NAD+存在的情况下，该反应是由预苯酸脱氢酶催化的。这种酶是至关重要的，因为它将酪氨酸和苯丙氨酸生物合成的中间分支点预苯酸引导到酪氨酸合成。酪氨酸的生物合成对肠道细菌、酵母、真菌和植物的生长和生存至关重要。与其他芳香族氨基酸一样，酪氨酸在生物体的生物化学中扮演着双重角色，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，而在后一种情况下，它是下游代谢途径中酶的底物。酪氨酸衍生的芳香族代谢物包括苯醌[2，3]、生氰苷[4]、生物碱[5，6]、黄酮类[7]和苯丙烷途径衍生的酚类化合物[7，8]。因为这些化合物中的许多都参与了主要的生物过程，所以它们对于生存是必不可少的。例如，在植物中，类黄酮对正常发育很重要，因为它们参与生长素的运输[9-11]，花粉萌发[9，12，13]，以及向共生微生物发出信号[9，14]。我们最近已经确定了预苯酸脱氢酶的晶体结构，但我们认为，我们对该酶机制的理解取决于确定其与已知配体的络合物的晶体结构。我们最近分别用预苯酸酯、(4-羟基苯基)丙酮酸和酪氨酸进行了共结晶研究，得到了预苯酸脱氢酶的晶体。我们正在请求X射线数据收集的束流时间，以确定与这些配体形成的络合物中的预苯酸脱氢酶的结构。 莽草酸脱氢酶：莽草酸途径参与细菌、植物、真菌和尖端复合体寄生虫体内芳香族氨基酸、叶酸、维生素、醌和各种其他芳香族化合物的生物合成。其中一些芳香族化合物对这些生物的生存是必不可少的；因此，莽草酸途径一直是抗菌和除草剂设计的一个有吸引力的目标。这种双功能酶脱氢奎尼酸脱水酶-莽草酸脱氢酶(DHQ-SDH)在莽草酸途径中催化脱氢奎尼酸脱水成脱氢莽草酸，然后将脱氢莽草酸还原成莽草酸。我们最近测定了DHQ-SDH的晶体结构。我们最近制作了一些活性位点突变体，这些突变对于我们理解这种酶的催化机制是很重要的。我们现在已经获得了这些突变变体的晶体，并正在请求用于X射线数据收集的光束时间。 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。其中一种机制是多重抗生素耐药(MAR)，在MAR中，微生物通过上调药物外排泵的表达来降低细胞内抗生素的浓度，药物外排泵积极地将类药物化合物从细胞中移除。我们最近确定了一种多重抗生素耐药抑制蛋白(MARR)的晶体结构，并与一些药物化合物进行了共结晶研究。我们在一些共结晶研究中获得了MARR的晶体，并要求获得用于X射线数据收集的光束时间。我们的目标是确定MARR与不同药物化合物的复合体的结构，这对于我们理解多重耐药的机制是很重要的。

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.