Mechanisms, Structures and Engineering of Flavonoid-producing Dioxygenase Enzymes - ABSTRACT The overall objective of this research is to fill in the gaps that exist in our structural and mechanistic understandings of alpha ketoglutarate-dependent dioxygenases, a medically important family of enzymes that perform a broad repertoire of biochemical transformations. The flavonoid biosynthetic pathway is dependent upon Flavanone Synthase 1 (FLS1) and Flavanone 3-Hydroxylase (F3H) to make medically significant products, quercetin and (2S,3R)-trans-taxifolin, respectively. These products have demonstrated health benefits and are broadly studied to better understand their cellular and metabolic roles in maintaining human health and the health of the gut microbiome. However, the mechanistic and structural understanding of the enzymes that make these key flavonoids is incomplete. A stronger biochemical understanding of these enzymes is needed to inform and diversify strategies to biosynthesize flavonoids that are useful for research. The first aim of this project is to use an innovative method to measure the mechanisms of FLS1 and F3H. This method will have broad applicability for studying the mechanisms of other enzymes in the family of alpha ketoglutarate-dependent dioxygenases. Techniques used to achieve this aim include high resolution mass spectrometry and biosynthesis of isotope labeled products. The second aim of this project is to elucidate the structures of FLS1 and F3H bound to products and substrates. Findings from the second aim will help to support the mechanistic findings of the related first aim. In addition, these findings will provide accurate models for future studies and enzyme engineering applications, with the goal of diversifying flavonoid biosynthetic strategies. The method used to achieve the second aim is primarily protein X-ray crystallography. The products of this research will increase our understanding of the enzyme mechanisms of alpha ketoglutarate-dependent dioxygenases as well as provide new methods to study them. In addition, new mechanistic and structural knowledge gained about the flavonoid synthesizing enzymes, FLS1 and F3H, will unlock strategies to create new biosynthetic tools for studying the effects and fates of their product metabolites, as they relate to human health and disease.
