Date of Defense

4-21-2023

Date of Graduation

6-2023

Department

Chemistry

First Advisor

Frederick Stull

Second Advisor

Todd Barkman

Abstract

6-hydroxy-(L)-nicotine oxidase, isolated from the organism Shinella sp. HZN7 (6HLNO-SE), is an enzyme that during the catabolism of nicotine catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudeooxynicotine. This enzyme is a member of the monoamine oxidase (MAO) structural family of flavoproteins, which contain a highly conserved FAD-binding domain. Within the binding domain is a conserved lysine residue that can be traced throughout evolutionary history in this family. 6HLNO isolated from Arthrobacter nicotinovorans serves as a model enzyme for the one isolated from Shinella as both catalyze the same reaction in this biochemical pathway and much has been studied about the Arthrobacter enzyme. However, the lysine residue in Shinella does not reside in the same location as it has throughout evolutionary history and these two enzymes share little common amino acid identity. This study aims to understand the mechanism of 6HLNO-SE as compared to that of Arthrobacter by using steady-state and transient kinetic assays via an O2 probe and stopped-flow experimentation to analyze mutagenesis effects and pH-dependence of the active site residues. Results demonstrate that the lysine residue as conserved throughout evolutionary history is critical for reactivity in 6HLNO-SE and that, though distantly related, the Shinella and Arthrobacter enzymes have very similar behavior.

Access Setting

Honors Thesis-Open Access

Additional Files
Honors Thesis Presentation.pdf (3384 kB)
Defense Presentation
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