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A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

ABSTRACT: Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O₂-dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O₂, severely limiting its nicotine-degrading activity. Instead of using O₂ as an oxidant, we discovered that NicA2 donates electrons to a cytochrome c, which means that NicA2 is actually a dehydrogenase. This is surprising, as enzymes of the flavin-containing amine oxidase family were invariably thought to use O₂ as an electron acceptor. Our findings establish new perspectives for engineering this potentially useful therapeutic and prompt a reconsideration of the term 'oxidase' in referring to members of the flavin-containing amine 'oxidase' family.

SUBMITTER: Dulchavsky M

PROVIDER: S-EPMC7904663 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

Dulchavsky Mark M Clark Christopher T CT Bardwell James C A JCA Stull Frederick F

Nature chemical biology 20210111 3

Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O<sub>2</sub>-dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O<sub>2</sub>, severely limiting its nicotine-degrading activit ...[more]

PMID: 33432238

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Project description:Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol · min-1 · mg of protein-1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.

Project description:Microbially-mediated uranium bioremediation has been demonstrated in uranium contaminated aquifers when acetate was artificially supplied and growth of the natural population of Geobacteraceae was stimulated. In order to mimic the environmental response to acetate, steady-state cells of G. sulfureducens were cultured in chemostats under conditions of either 1) acetate as the sole electron donor and limiting factor and fumarate as the sole electron acceptor or 2) acetate was supplied in excess with fumarate as sole electron acceptor and limiting factor. In silico fluxome modeling and transcriptome analysis were used as tools for investigating the cell response to the acetate availability. For global gene expression profiling, a DNA microarray of the complete G. sulfurreducens genome was used. Statistically significant results were obtained from two-color, dye swap hybridizations produced from a total of three biological replicates. Eight technical replicates were tested from two of the biological replicates and six technical replicates were tested from the third biological replicate. Major findings from this study are given as follows. The in silico model successfully predicted a higher TCA-cycle flux (ca. 2-fold) under acetate-excess conditions, suggesting that catabolism of acetate is favored with respect to anabolism, and thus more electrons are available for metal reduction. Transcriptome analyses offered a comprehensive picture of the regulation points subjected to the acetate availability. Under acetate-excess conditions, acetate transporters in the G. sulfurreducens genome were down-regulated. In addition the oxidation-related acetyl-CoA transferase was up-regulated approximately three-fold and the assimilatory-related acetate kinase was down-regulated approximately two-fold, respectively, indicating that the transcriptional regulation of acetate activation may be the key point for coping with the excess of acetate and increasing the TCA flux. The level of transcription for 10 c-type cytochromes was significantly increased in cells cultured with an excess of acetate. OmcS, an outer-membrane cytochrome which actively participates in electron transfer to Fe(III)-oxides and graphite electrodes from fuel cells, showed one of the highest fold increases in transcription. The integration of in silico modeling and genome-wide analysis shows for first time how G. sulffureducens adapts its metabolic flux and transcriptional network for optimizing the use of acetate as an electron donor for exocellular respiration instead of for use as a carbon source for biomass production. Keywords: Geobacter, gene expression, acetate limitation, fumarate limitation

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A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

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A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

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