Date of Defense

4-25-2022

Date of Graduation

4-2022

Department

Chemistry

First Advisor

Frederick Stull

Second Advisor

Todd Barkman

Abstract

Nicotine oxidoreductase (NicA2) found in the organism Pseudomonas putida S16 is capable of the degradation of nicotine into non-addictive metabolites. It has recently been discovered that NicA2 through its degradation of nicotine donates it electrons from this redox reaction to a cytochrome c (CycN) protein. This study attempts to identify the binding interface between these two proteins using uncommon amino acid incorporation. The photocrosslinking, uncommon amino acid p-benzoyl-L-phenylalanine (Bpa) was incorporated at several residues on the surface of NicA2 in an attempt to generate a crosslinked species of NicA2 and CycN. A crosslinked species was not able to be generated and hence other means of kinetic analyses were utilized. Due to the steric properties of Bpa, a hypothesis was formed that the Bpa containing NicA2 mutants could potentially interfere with the binding of NicA2 to CycN, if Bpa was incorporated at a residue of NicA2 that is important for binding. A steady-state assay showed this to be true for four residue locations F93, F104, D295, and R393. Stopped-flow experiments analyzing both the first redox reaction between NicA2 and nicotine and the second redox reaction between NicA2 and CycN verified this and showed that Bpa incorporation at these sites drastically reduced the rate of the reaction between NicA2 and CycN but not with nicotine oxidation.

Access Setting

Honors Thesis-Restricted

Restricted to Campus until

6-24-2023

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