An NAD(P)H-Nicotine Blue Oxidoreductase Is Part of the Nicotine Regulon and May Protect Arthrobacter nicotinovorans from Oxidative Stress during Nicotine Catabolism

Abstract

An NAD(P)H-nicotine blue (quinone) oxidoreductase was discovered as a member of the nicotine catabolic pathway of Arthrobacter nicotinovorans. Transcriptional analysis and electromobility shift assays showed that the enzyme gene was expressed in a nicotine-dependent manner under the control of the transcriptional activator PmfR and thus was part of the nicotine regulon of A. nicotinovorans. The flavin mononucleotide-containing enzyme uses NADH and, with lower efficiency, NADPH to reduce, by a twoelectron transfer, nicotine blue to the nicotine blue leuco form (hydroquinone). Besides nicotine blue, several other quinones were reduced by the enzyme. The NAD(P)H-nicotine blue oxidoreductase may prevent intracellular one-electron reductions of nicotine blue which may lead to semiquinone radicals and potentially toxic reactive oxygen species.The unraveling of the details of nicotine catabolism in the model organism Arthrobacter nicotinovorans stays in the center of our research efforts. The final goal is to obtain a comprehensive picture of the biochemical steps implicated in this process (7) and to find ways for their ultimate use in biotechnological applications.When grown on nicotine, A. nicotinovorans cultures turn dark blue due to the formation of a pigment known as nicotine blue (NB). Chemically, NB was shown to be a 4,5,4Ј,5Ј-tetrahydroxy-3,3Ј-diazadiphenoquinone-(2,2Ј) formed by the autocatalytic condensation of two molecules of trihydroxypyridine (THP) (18,20). The steps in nicotine catabolism leading to THP (Fig. 1) start with the cleaving of 2,6-dihydroxypseudooxynicotine into 2,6-dihydroxypyridine (DHP) and ␥-N-methylaminobutyrate by the enzyme 2,6-dihydroxypseudooxynicotine hydrolase (PONH) (26). ␥-N-methylaminobutyrate is converted to the citric acid cycle intermediate succinate by the enzymes ␥-N-methylaminobutyrate oxidase (MABO) (11), monoamine oxidase, and succinate semialdehyde dehydrogenase (13). DHP is hydroxylated to 5-hydroxy-2,6-(1H,3H)-pyridinedione by the flavoenzyme DHP hydroxylase (4). The enzyme reaction can be detected in vitro by monitoring at 578 nm the spontaneous formation of nicotine blue from THP.The gene for MABO forms one operon with the genes for the enzymes formyltetrahydrofolate deformylase (purU) and methylentetrahydrofolate dehydrogenase-cyclohydrolase (folD), which are required for the metabolism of methylenetetrahydrofolate formed during the oxidative demethylation of ␥-N-methylaminobutyrate (11, 13) and the genes for a small, two-component multidrug resistance pump (SmrAB) (P. Ganas and R. Brandsch, unpublished data). The expression of this operon is regulated by the transcriptional activator PmfR (12). The genes for the monoamine oxidase (maO) and succinate semialdehyde dehydrogenase (ssD) are divergently transcribed (13). The gene cluster is located on the catabolic megaplasmid pAO1 (19) of A. nicotinovorans, flanked by a transposon similar to Tn554 and the truncated gene for a transposase (Fig. 2). The protein derived from orf481 exhibits a high degree of similarity to...

Time-Dependent Analysis of Paenarthrobacter nicotinovorans pAO1 Nicotine-Related Proteome

“…The key enzymes processing the resulting intermediates and performing the last steps of the pathway are significantly more abundant in the late stationary phase. Two of these enzymes, the 2-oxoglutaramate amidase (NIT) and the hypothetical polyketide cyclase (PKC), are believed to be involved in the cleavage of the THP ring, while the NAD­(P)­H-nicotine blue oxidoreductase (NBOR) is believed to be involved in maintaining the NB pigment in an oxidation stage favorable for cleavage. , The abundance of these enzymes in the late stationary phase when nicotine could not be detected in the medium, and, thus should not further activate the transcription of the nic -genes, could be related to the decrease in the NB pigment levels observed in Figure A. Still, we are hesitant to conclude that this is a clear indication that an NB degradation takes place and that these are the enzymes involved.…”

“…The known end products of the nicotine degradation in P. nicotinovorans pAO1 are methylamine, succinic acid, and NB. Both NB and methylamine are excreted into the medium. , Nevertheless, several reports indicate that NB is actually reimported into the cell, reduced by a NAD­(P)­H-NBOR, and slowly converted into alpha-ketoglutarate. , Hence, both the end products succinic acid and alpha-ketoglutarate should be integrated into the general pathways of the cells and used for growth.…”

Proteomics based analysis of the nicotine catabolism in Paenarthrobacter nicotinovorans pAO1

“…It was postulated that this oxidation reaction may represent a selective advantage for P . nicotinovorans bacteria in competition with other soil community bacteria sensitive to oxygen radicals 21 and that the NAD(P)H-Nicotine Blue Oxidoreductase (NBOR) enzyme might prevent the formation of these radicals inside the cell. Our MS data indicate that the mechanism by which P .…”

Dynamic metabolomic responses of Escherichia coli to nicotine stress

“…We suppose that this model bacterium should present a distinctive physiological response to nicotine stress for survival. However, most studies to date have mainly focused on investigating the alterations of gene expression and protein in cells or microorganisms exposed to nicotine stress (Crowley-Weber et al 2003;Mihasan et al 2007;Rothem et al 2009;Baek et al 2012). The metabolic mechanisms by bacteria in response to nicotine stress remain to be identified.…”