New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of γ‐N‐methylaminobutyrate (CH3‐4‐aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH3‐4‐aminobutyrate could be identified. CH3‐4‐aminobutyrate may be demethylated to γ‐N‐aminobutyrate by the recently identified γ‐N‐methylaminobutyrate oxidase [Chiribau et al. (2004) Eur J Biochem271, 4677–4684]. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH3‐4‐aminobutyrate with the formation of succinic semialdehyde. Succinic semialdehyde was converted to succinate by a NADP+‐dependent succinic semialdehyde dehydrogenase. Succinate may enter the citric acid cycle completing the catabolism of the pyrrolidine moiety of nicotine. Expression of the genes of these enzymes was dependent on the presence of nicotine in the growth medium. Thus, two enzymes of the nicotine regulon, γ‐N‐methylaminobutyrate oxidase and amine oxidase share the same substrate. The Km of 2.5 mm and kcat of 1230 s−1 for amine oxidase vs. Km of 140 µm and kcat of 800 s−1 for γ‐N‐methylaminobutyrate oxidase, determined in vitro with the purified recombinant enzymes, may suggest that demethylation predominates over deamination of CH3‐4‐aminobutyrate. However, bacteria grown on [14C]nicotine secreted [14C]methylamine into the medium, indicating that the pathway to succinate is active in vivo.

The first inducible Arthrobacter overexpression system, based on the promoter/operator and the repressor of the 6-D-hydroxynicotine oxidase gene of Arthrobacter nicotinovorans, is described here. Nicotine-dependent overproduction and affinity purification of recombinant proteins are presented. The system will allow the production of complex enzymes and genetic complementation studies in Arthrobacter species.

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...