MECHANISTIC STUDIES OF ELONGATOR PROTEIN 3 AND PERIPLASMIC NITRATE REDUCTASE

Abstract

Bioinorganic chemistry is an important field of chemistry that strives to understand the roles of metalloproteins and the chemistry they catalyze. Herin, I describe my efforts to characterize the mechanism of two metalloenzymes, Elongator Protein 3 (Elp3) and Periplasmic Nitrate Reductase (NapA). Elp3 is a member of the Radical SAM superfamily of enzymes. The enzyme contains two domains, the Radical SAM and Lysine Acetyl Transferase (KAT) domain. The preferential substrate for Elp3 is aminoacyl tRNAs that harbor a wobble uridine at position 34. Elp3 modifies the wobble uridine through the addition of a carboxymethyl group derived from Acetyl-CoA. Evidence for the Elp3 mechanism is lacking. It has been hypothesized that reaction is facilitated through a radical reaction that abstracts an H-atom generating an Acetyl radical which adds to the C-5 position of uridine to create 5-carboxymethyluridine (cm5U). This is a challenge given the domains are ~40 Å apart. I have investigated possible mechanisms by which this may occur using a bacterial homologue of Elp3 (Dehalococcoides mccartyi; DmElp3). NapA is part of the DMSO Reductase family of molybdopterin enzymes and catalyzes the reduction of nitrate to nitrite at the molybdenum center through an oxygen atom transfer reaction. Despite extensive research on molybdenum enzymes, including NapA, there are significant questions regarding the coordination environment of the molybdenum and the resulting mechanism of oxygen atom transfer. Here, I describe my efforts to characterize the mechanism and influence of the primary and secondary coordination sphere on catalysis. I describe the importance of a highly conserved lysine in the secondary coordination sphere as well as the likely route of inhibition by cyanide. Rapid reaction kinetics of the wildtype enzyme has shed light on the mechanism revealing that the steady state turnover is rate limited by reduction of the enzyme, not the chemistry of oxygen atom transfer. The results of both Elp3 and NapA are discussed in the context of the proposed mechanisms for the respective enzymes.