The Biochemistry of 4-Hydroxyphenylpyruvate

本研究的首要目标是了解4-羟基苯基丙酮酸酯（HPP）的生物化学。HPP是一种非常通用的次级代谢物，在每个生命王国中都具有保守和独特的作用。在这个项目的化学两个迷人的相似的-酮酸依赖酶的共同祖先进行比较。这些酶表面上具有相同的整体结构，都使用HPP和氧作为反应物，但却能形成完全不同的产物。这些酶是4-羟基苯基丙酮酸双加氧酶（HPPD）和羟基曼德酸合成酶（HMS）。-酮酸依赖酶是已知的最大和最多样化的加氧酶。这个家族的酶催化一系列复杂的反应，这些反应与许多不同的生化过程有关，从允许进入高等生物的DNA到植物毒素的合成或胶原蛋白的形成。关于这些酶催化过程中发生的步骤的广泛猜测在文献中持续存在，但在很大程度上没有证据支持。这种综合的方法采用了光谱学、晶体学和诱变学，以提供结构-功能关系的详细图像。首席研究员的实验室已经证明，在HPPD和HMS的反应中，至少有四种独特的化学物质发生。显然，证明这些中间产物身份的证据将是一个非常重要的观察结果。此外，反应物和产物配合物的结构是对任何一种酶进行的关键观察之一，并将为每种反应提供结构背景。因此，本研究的主要目标是捕获和表征高活性中间体，以了解这两种反应途径的基础，获得与反应物和产物结合的酶的附加结构，并研究特异性突变的酶，以探测这两种化学反应的主要决定因素。这项研究的成功完成将对氧气利用的理解产生重大影响，并将提供对高活性物种如何由加氧酶指导的见解。更广泛的影响。HPPD和HMS项目已被证明是整合教学和研究的特别有用的平台。这些酶化学的丰富性和广泛相关性意味着从事这项研究的学生可以接触到各种各样的方法和科学猜想，这些方法和猜想使他们的工作充满好奇心和使命感。首席研究员利用该研究项目的数据继续改进完全基于HPPD生物化学的本科实验课程。此外，威斯康星大学密尔沃基分校的使命是教育来自周围城市社区的代表性不足的学生。因此，首席研究员可以很好地接收来自不同背景和遗产的学生，最近的博士和学士毕业生证明了这一点。此外，首席研究员通过每年提供他的专业知识来评判高中科学博览会，从而参与当地的科学推广，这些高中科学博览会不断产生国家科学博览会的竞争者。

The overarching goal of this research is to understand the biochemistry of 4-hydroxyphenylpyruvate (HPP). HPP is a remarkably versatile secondary metabolite that has both conserved and unique roles in each Kingdom of life. In this project the chemistries of two fascinatingly similar alpha-keto acid dependent enzymes of common ancestry are compared. These enzymes have ostensibly the same overall structure and both use HPP and oxygen as reactants but manage to form entirely different products. These enzymes are 4-hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS). The alpha-keto acid dependent enzymes are the largest and most diverse set of oxygenase enzymes known. Enzymes in this family catalyze an array of complex reactions that are relevant to many diverse biochemical processes, from permitting access to DNA in higher organisms to the synthesis of plant toxins or the formation of collagen. Widespread speculation as to the steps that occur during catalysis by these enzymes persists in the literature but is largely unsupported by evidence. This comprehensive approach employs spectroscopy, crystallography, and mutagenesis to allow for a detailed picture of structure-function relationships to emerge. The laboratory of the principal investigator has shown that at least four unique chemical species occur during reactions of HPPD and HMS. Clearly, evidence for the identity of these intermediates would be a highly significant observation. In addition, the structure of both the reactant and product complexes is one of the key observations yet to be made for either enzyme and will provide a structural context for each reaction. As such, the broad objectives of this research are to trap and characterize highly reactive intermediates in order to understand the basis for these two reaction paths, obtain additional structures of both enzymes bound to reactants and products and to investigate specifically mutated enzymes to probe for the primary determinants of these two chemistries. The successful completion of this research will have significant impact on the understanding of oxygen utilization and will also provide insight into how highly reactive species are directed by oxygenase enzymes. Broader Impact. The HPPD and HMS projects have proven to be particularly useful platforms for the integration of teaching and research. The richness and broad relevance of the chemistry of these enzymes has meant that students who undertake this research gain exposure to a wide array of methodologies and scientific conjecture that drives their work from a sense of both curiosity and purpose. The principal investigator uses the data from this research program to continue to improve an undergraduate laboratory course based entirely on the biochemistry of HPPD. Moreover, The University of Wisconsin- Milwaukee has a mission to educate underrepresented students from surrounding urban communities. As such the principal investigator is well placed to receive students from diverse backgrounds and heritage as evidenced by recent doctoral and bachelors graduates. Moreover, the principal investigator is involved in the promotion of science locally by offering his expertise each year to judge high school science fairs that continue to yield national science fair competitors.