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Publication Abstract from PubMed

The soil-dwelling bacterium Pseudomonas putida S16 can survive on nicotine as its sole carbon and nitrogen source. The enzymes nicotine oxidoreductase (NicA2) and pseudooxynicotine amine oxidase (Pnao), both members of the flavin containing amine oxidase family, catalyze the first two steps in the nicotine catabolism pathway. Our laboratory has previously shown that, contrary to other members of its enzyme family, NicA2 is actually a dehydrogenase that uses a cytochrome c protein (CycN) as its electron acceptor. The natural electron acceptor for Pnao is unknown; however, within the P. putida S16 genome, pnao forms an operon with cycN and nicA2, leading us to hypothesize that Pnao may also be a dehydrogenase that uses CycN as its electron acceptor. Here we characterized the kinetic properties of Pnao and show that Pnao is poorly oxidized by O2, but can be rapidly oxidized by CycN, indicating that Pnao indeed acts as a dehydrogenase that uses CycN as its oxidant. Comparing steady-state kinetics with transient kinetic experiments revealed that product release primarily limits turnover by Pnao. We also resolved the crystal structure of Pnao at 2.60 A, which shows that Pnao has a similar structural fold as NicA2. Furthermore, rigid-body docking of the structure of CycN with Pnao and NicA2 identified a potential conserved binding site for CycN on these two enzymes. Taken together, our results demonstrate that although Pnao and NicA2 show a high degree of similarity to flavin containing amine oxidases that use dioxygen directly, both enzymes are actually dehydrogenases.

The enzyme pseudooxynicotine amine oxidase from Pseudomonas putida S16 is not an oxidase, but a dehydrogenase.,Choudhary V, Wu K, Zhang Z, Dulchavsky M, Barkman T, Bardwell JCA, Stull F J Biol Chem. 2022 Jul 11:102251. doi: 10.1016/j.jbc.2022.102251. PMID:35835223^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.