Rapid Reaction Studies on the Chemistry of Flavin Oxidation in Urocanate Reductase

Date of Award


Degree Name

Master of Science



First Advisor

Fredrick Stull, Ph.D.

Second Advisor

David Huffman, Ph.D.

Third Advisor

Todd J Barkman, Ph.D.

Access Setting

Masters Thesis-Abstract Only

Restricted to Campus until



Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and the metabolic disorder insulin resistance. Here we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA’) using anaerobic stopped-flow experiments at 4 °C. This analysis unveiled the presence of a charge transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (~1 ms). The flavin of this intermediate was then oxidized by urocanate with a rate constant of ~60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA’ are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Furthermore, the mutational analysis of urocanate binding residues in UrdA’ suggests that the twisted conformation of urocanate imposed by the active site of UrdA’ facilitates urocanate reduction by the enzyme.

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