Date of Award

8-2022

Degree Name

Master of Science

Department

Chemistry

First Advisor

Frederick Stull, Ph.D.

Second Advisor

David Huffman, Ph.D.

Third Advisor

Todd Barkman, Ph.D.

Keywords

Crystallography, enzymology, flavin containing enzymes, nicotine catabolism, pnao, pseudomonas putida S16

Access Setting

Masters Thesis-Open Access

Abstract

Our laboratory has previously shown that nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, uses a cytochrome c protein (CycN) as its electron acceptor rather than characteristically accepted molecular oxygen. Within the Pseudomonas putida S16 genome, the nicA2 gene forms an operon with cycN and pseudooxynicotine amine oxidase (pnao), another flavin containing amine oxidase enzyme. The first two steps in the nicotine catabolism pathway are catabolized by NicA2 and Pnao. Through redox chemistry, NicA2 converts nicotine to pseudooxynicotine and Pnao converts pseudooxynicotine to 3-succinoylsemialdehyde-pyridine. In this thesis, we characterize the kinetic and structural properties of Pnao. Similar to NicA2, Pnao is poorly oxidized by O2 but can rapidly reduce CycN, contradicting the largely conserved “oxidase” behavior within the flavin-dependent amine oxidase family of enzymes. We also observed that, like NicA2, Pnao is unable to reduce other cytochrome c proteins, and resolved the crystal structure for Pnao at 2.60 Å, which reveals that, despite only sharing ~40% sequence identity, Pnao has a similar overall structural fold as NicA2. Our results indicate that, despite having similarities to flavin containing amine oxidases, Pnao, like NicA2, should be reclassified as a dehydrogenase.

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