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.
" (award number MCB-0843619). The amount of funding was $532,012 (direct + indirect) and spanned the period April 1st 2009 through March 31st 2012 (no cost extension for 12 months through March 31st 2013). The primary research output includes ten articles. Intellectual Merit: Three are invited review articles that describe mechanistic and structural aspects of 4-Hydroxyphenylpyruvate dioxygenase (HPPD) and the sister enzyme hydroxymandelate synthase (HMS). The remaining seven articles are research based and describe significant advances in our understanding of both enzymes. Two publications were collaborative with the laboratory of Edward Solomon from Stanford University and focus on the early parts of the chemical mechanism of HPPD. The remaining five articles detail (a) an expansion of our knowledge of the catalytic steps in HMS, (b) the importance of a conserved asparagine residue to catalysis, (c) the catalytic consequence of a specific mutation of this asparagine residue that is causative for the disease Hawkinsinuria, (d) a proposed remedy for this condition in the form of a drug (that was developed as a herbicide) that is used for other rare defects in tyrosine catabolism and (e) major advancements in our understanding of the hydroxylation reactions of both HPPD and HMS. Each of these publications are listed below. Broader Impacts: Three graduate students and three postdoctoral researchers were supported in the prior funding period.Graduate student, Panqing He has entered employment in the oil industry in California are completing postdoctoral studies at University of Washington with Dr. Wesley van Voorhis. Graduate student, Judith Bates, has become a lecturer at Milwaukee Area Technical College. Graduate student, Dhara Shah will graduate in the Spring of 2014. Posdoctoral Associate, John Conrad is currently an Assistant Professor at the University of Nebraska Omaha Department of Chemistry.
