Final steps in the catabolism of nicotine

Abstract

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.

Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

“…Nicotine was hydroxylated at position 6 of the pyridine ring, and the pyrrolidine ring was later cleaved. Comparing the two different degradation processes, the corresponding steps catalyzed by PNAO and SAPD in strain HZN6 were the deamination of ␥-N-methylaminobutyrate by an amine oxidase (AO) and the dehydrogenation of succinic semialdehyde by succinic semialdehyde dehydrogenase (SsaDH) in A. nicotinovorans pAO1, respectively (6). Similar to the AO of pAO1, the FAD cofactor of PNAO was noncovalently bound to the apoprotein, and the PNAO activity was also substrate specific.…”

“…The secondary-aminecontaining compound was the product of 6HLNO while serving as a substrate of PNAO. In addition, similar to SsaDH, the SAPD activity was NADP ϩ dependent and not substrate specific (6). Because the PNAO showed no activity to nicotine, according to the nicotine-degrading mechanism of Arthrobacter nicotinovorans pAO1 (5, 6), we can infer that the initial steps of nicotine degradation are catalyzed by the products of three individual genes in Pseudomonas sp.…”

“…The methylamine produced in the enzyme assay with PNAO was identified using a thin-layer chromatography (TLC) method similar to that of Chiribau, with silica gel HF254 and n-butanol-acetone-acetic acid-H 2 O (10:15:3:12 [vol/vol/vol/vol]) as the mobile phase (6). The plates were developed by spraying with a 0.1% (vol/vol) ninhydrin solution in acetone.…”

“…In the Gram-positive strain Arthrobacter nicotinovorans, the degradation pathway (the pyridine pathway) and the related genes and enzymes have been well elucidated (2,(5)(6)(7)(10)(11)(12). Although aerobic nicotine degradation has been studied in different Gram-negative Pseudomonas strains, the genes encoding enzymes in the pyrrolidine pathway involved in nicotine degradation have yet to be analyzed in detail (16).…”

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

“…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).…”