Project Summary/Abstract 2-Oxoglutarate (2OG) dependent nonheme mononuclear iron (NHM-Fe) enzymes catalyze an exceedingly broad scope of reactions that are involved in key chemical transformations of many important biological pathways, such as gene regulation, epigenetics, and natural product biosynthesis. Although detailed mechanistic understandings of the canonical hydroxylation reactivity found in 2OG/NHM-Fe enzymes have been developed in recent years, it remains unknown how this hydroxylation paradigm can fully explain non- hydroxylation reactivity in this family of enzymes, such as desaturation and epoxidation. Furthermore, given the catalytic abilities of 2OG/NHM-Fe enzymes to construct pharmaceutically valuable molecular scaffolds, exploiting these enzymes for biocatalysis applications represents an attractive but under developed area for expanding natural product based compound libraries. In this proposal, we seek to provide critical improvements on these under developed areas through the studies of AsqJ, a novel multifunctional 2OG/NHM- Fe enzyme that is involved in Viridicatin-type quinolone alkaloid biosynthesis in Aspergillus nidulans. AsqJ catalyzes a chemically interesting sequential desaturation/epoxidation reaction to construct Viridicatin core structure, which represents a chemically unexplored strategy for Viridicatin synthesis. A multi-faceted experimental method will be utilized to elucidate AsqJ reaction mechanisms, which consists of organic synthesis, molecular cloning, biochemical assays, protein crystallography, pre-steady state kinetics, and advanced spectroscopic techniques. This method will be further supplemented with molecular dynamic simulations to generate molecular level understandings of the AsqJ catalysis. It is expected that the proposed research will provide critical improvements to the mechanistic understandings of desaturation and epoxidation, two chemically challenging but under explored reactions catalyzed by 2OG/NHM-Fe enzymes, and further explore mechanism based bioengineering approach to access viridicatin-type scaffolds.