6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33

Abstract

Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O 2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P) ϩ , O 2 , and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoylsemialdehyde-pyridine was verified to be derived from H 2 O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria. IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O 2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

Physiology of a Hybrid Pathway for Nicotine Catabolism in Bacteria

“…The intermediate 6-hydroxy-pseudooxynicotine is further dehydrogenated into 6-hydroxy-3-succinoyl-semialdehyde-pyridine by 6-hydroxy-pseudooxynicotine dehydrogenase (Pno), which was formerly thought to be an oxidase since it catalyzes a reaction similar to that by pseudooxynicotine amine oxidase (Pao or Pnao) from Pseudomonas sp. HZN6 and P. putida S16 ( Li et al, 2016 ; Wang et al, 2019a ). By coupling with the reaction of 6-hydroxynicotine oxidation by Hno, the activity of Pno at 32.3 U/mg and an apparent K m of 0.37 mM for 6-hydroxy-pseudooxynicotine were estimated at pH 8.5 and 30°C.…”

“…NdhAB utilizes Paz as its electron acceptor, which can further deliver the electrons to cytochrome c and then to the final electron acceptor O 2 catalyzed by cytochrome c oxidase, or directly to O 2 considering the pseudospecificity between Paz and cytochrome c ( Yu et al, 2017a ). Pno transfers the electron to EtfAB, and then to CoQ catalyzed by Euo, which enters the classic electron transport chain (ETC; Wang et al, 2019a ). Ald uses NAD(P) as an electron acceptor, and the reduced equivalent can be reoxidized when it takes part in the reaction of Hsh.…”

Structural and biochemical characterization of iminodiacetate oxidase from Chelativorans sp. BNC1

“…This observation may reflect the fact that IdaA activity is seven times slower when it uses O 2 instead of an artificial electron acceptor ferricyanide (Liu et al , ). It is plausible that IdaA passes the reducing power to coenzyme Q via the electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxidoreductase (Zhang et al , ), as 6‐hydroxypseudooxynicotine dehydrogenase of Agrobacterium tumefaciens S33 does (Wang et al , ).…”

An NAD-Specific 6-Hydroxy-3-Succinoyl-Semialdehyde-Pyridine Dehydrogenase from Nicotine-Degrading Agrobacterium tumefaciens Strain S33

“…Next, the hybrid pathway follows the pyrrolidine pathway through HSP and 2,5-dihydroxypyridine, and finally, it enters the tricarboxylic acid (TCA) cycle. To clarify the biochemical mechanism of this special hybrid pathway, great efforts have been put into studies in recent years, including the analysis of its genome and transcriptome and the purification and characterization of its key enzymes ( 6 , 12 , 15 – 21 ). Now, a better understanding of the hybrid pathway has been achieved ( 22 ) after the key oxidoreductases involved in the pathway, including nicotine dehydrogenase (NdhAB), 6-hydroxynicotine oxidase (Hno), 6-hydroxypseudooxynicotine dehydrogenase (Pno), reactive imine deaminase (Rid), and 6-hydroxy-3-succinoylpyridine hydroxylase (Hsh), were purified and characterized ( Fig.…”

“…Now, a better understanding of the hybrid pathway has been achieved ( 22 ) after the key oxidoreductases involved in the pathway, including nicotine dehydrogenase (NdhAB), 6-hydroxynicotine oxidase (Hno), 6-hydroxypseudooxynicotine dehydrogenase (Pno), reactive imine deaminase (Rid), and 6-hydroxy-3-succinoylpyridine hydroxylase (Hsh), were purified and characterized ( Fig. 1A ) ( 15 – 18 , 20 , 21 ). However, there are still some unresolved problems; one of which is that the enzyme responsible for converting 6-hydroxy-3-succinoyl-semialdehyde-pyridine to HSP has not yet been identified.…”