Final steps in the catabolism of nicotine

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 (CH 3 ‐4‐aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH 3 ‐4‐aminobutyrate could be identified. CH 3 ‐4‐aminobutyrate may be demethylated to γ‐ N ‐aminobutyrate by the recently identified γ‐ N ‐methylaminobutyrate oxidase [Chiribau et al . (2004) Eur J Biochem 271, 4677–4684]. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH 3 ‐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 K m of 2.5 m m and k cat of 1230 s −1 for amine oxidase vs. K m of 140 µ m and k cat 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 CH 3 ‐4‐aminobutyrate. However, bacteria grown on [ 14 C]nicotine secreted [ 14 C]methylamine into the medium, indicating that the pathway to succinate is active in vivo .