Tài liệu Báo cáo khoa học: Final steps in the catabolism of nicotine Deamination versus

Final steps in the catabolism of nicotine

Deamination versus demethylation of c-N-methylaminobutyrate

Calin-Bogdan Chiribau1

, Marius Mihasan1,2, Petra Ganas1

, Gabor L. Igloi3

, Vlad Artenie2

and Roderich Brandsch1

1 Institute of Biochemistry and Molecular Biology, Alberts-Ludwig University of Freiburg, Germany

2 Department of Biochemistry, Alexandru Ioan-Cuza University of Iasi, Romania

3 Institute of Biology III, Alberts-Ludwig University of Freiburg, Germany

One of the major health risks continues to be the smok￾ing of tobacco. Nicotine, in itself highly toxic, when

inhaled with the tobacco smoke readily crosses the

blood–brain barrier. Its effects on the central nervous

system, mediated by cholinergic receptors, make it

highly addictive. As a result of nicotine addiction, only

a small percentage of smokers give up smoking [1]. In

addition, exposure to tobacco smoke in public places,

so-called secondary smoking, or to solid or liquid

waste during processing of tobacco products, repre￾sents a serious health threat. Therefore detoxification

of these tobacco waste products by removal of nicotine

is a major challenge. Several soil microorganisms have

evolved the enzymatic ability to mineralize nicotine,

Keywords

amine oxidase; Arthrobacter nicotinovorans;

nicotine; c-N-methylaminobutyrate; succinic

semialdehyde dehydrogenase

Correspondence

R. Brandsch, Institut fu¨r Biochemie und

Molekularbiologie, Hermann-Herder-Str. 7,

D-79104 Freiburg, Germany

Fax: +41 761 2035253

Tel: +41 761 2035231

E-mail: roderich.brandsch@biochemie.

uni-freiburg.de

(Received 23 November 2005, revised 1

February 2006, accepted 10 February 2006)

doi:10.1111/j.1742-4658.2006.05173.x

New enzymes of nicotine catabolism instrumental in the detoxification of

the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been iden￾tified and characterized. Nicotine breakdown leads to the formation of

nicotine blue from the hydroxylated pyridine ring and of c-N-methyl￾aminobutyrate (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-aminobuty￾rate may be demethylated to c-N-aminobutyrate by the recently identified

c-N-methylaminobutyrate oxidase [Chiribau et al. (2004) Eur J Biochem

271, 4677–4684]. In an alternative pathway, an amine oxidase with noncov￾alently 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,

c-N-methylaminobutyrate oxidase and amine oxidase share the same sub￾strate. The Km of 2.5 mm and kcat of 1230 s)1 for amine oxidase vs. Km of

140 lm and kcat of 800 s)1 for c-N-methylaminobutyrate oxidase, deter￾mined 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.

Abbreviations

AO, amine oxidase; CH3-4-aminobutyrate, c-N-methylaminobutyrate; CH2TH4, methylenetetrahydrofolate; DHPONH, dihydroxypseudo￾oxynicotine hydrolase; MABO, c-N-methylaminobutyrate oxidase; MAO, monoamine oxidase; TCA, trichloroacetic acid; TLC, thin layer

chromatography; SsaDH, succinic semialdehyde dehydrogenase.

1528 FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS