Final steps in the catabolism of nicotine Deamination versus demethylation of c-N-methylaminobutyrate Calin-Bogdan Chiribau 1 , Marius Mihasan 1,2 , Petra Ganas 1 , Gabor L. Igloi 3 , Vlad Artenie 2 and Roderich Brandsch 1 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 (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-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 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, c-N-methylaminobutyrate oxidase and amine oxidase share the same sub- strate. The K m of 2.5 mm and k cat of 1230 s )1 for amine oxidase vs. K m of 140 lm and k cat 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 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. Abbreviations AO, amine oxidase; CH 3 -4-aminobutyrate, c-N-methylaminobutyrate; CH 2 TH 4 , 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 but only the enzymes of nicotine catabolism of Arthrobacter nicotinovorans pAO1 have been character- ized into some detail [2]. Knowledge of the enzymes involved in nicotine catabolism will have applications not only in the bioremediation of nicotine waste, but also in the supply of nicotine derivatives as starting materials for the synthesis of new products of indus- trial and pharmaceutical importance [3,4]. Construc- tion of inducible mammalian systems responsive to nicotine and nicotine metabolites are feasible [5]. To achieve such goals, an in-depth understanding of the enzymology of nicotine catabolism is required. Our effort is directed towards the comprehensive characterization of the metabolic pathways of nicotine breakdown as it is present in the Gram-positive soil bacterium A. nicotinovorans [6]. A key step in the breakdown of nicotine by A. nicotinovorans carrying the catabolic plasmid pAO1 is the cleavage of 2,6-di- hydroxypseudooxynicotine into 2,6-dihydroxypyridine and c-N-methylaminobutyrate (CH 3 -4-aminobuty- rate) by 2,6-dihydroxypseudooxynicotine hydrolase (DHPONH, Fig. 1). This reaction is performed by a C–C bond hydrolase of the a ⁄ b fold family, the first shown to act on a heteroaromatic compound [7]. We have recently shown that a gene cluster on pAO1 is involved in the demethylation of CH 3 -4-aminobuty- rate. It consists of mabO, encoding the enzyme c-N- methylaminobutyrate oxidase (MABO, Fig. 1), which oxidatively demethylates CH 3 -4-aminobutyrate. This gene is flanked by a purU-like gene encoding a putative formyltetrahydrofolate deformylase and by a folD-like gene, encoding the putative bifunctional enzyme meth- ylenetetrahydrofolate (CH 2 TH 4 ) dehydrogenase-cyclo- hydrolase [8]. Expression of the purU-mabO-folD operon is regulated by the transcriptional activator PmfR and depends on the presence of nicotine in the growth medium [9]. Catabolism of 4-aminobutyrate produced in the MABO reaction could also proceed by oxidative deam- ination yielding succinic semialdehyde (Fig. 1, MAO broken arrow). A succinate semialdehyde dehydroge- nase (SsaDH, Fig. 1) would then channel the succinate formed in the reaction into the citric acid cycle. Indeed, next to the purU-mabO-folD operon there is on pAO1 a gabD-like gene (sad), encoding an SsaDH protein and a mao-like gene, encoding an amine oxid- ase (AO) (Fig. 2). In this work, we show that expression of these genes depends on the presence of nicotine in the growth medium and we have determined the enzyme activities of the proteins. Our results demonstrate the presence of two pathways of CH 3 -4-aminobutyrate catabolism, one yielding 4-aminobutyrate by oxidative demethyla- tion through MABO [8], and the other, by unexpected new enzyme specificity, yielding succinic semialdehyde by removing methylamine in an oxidative deamination reaction catalyzed by the AO (Fig. 1). Succinic acid semialdehyde is then converted to succinate by the SsaDH encoded by the sad gene of pAO1 (see Fig. 2). Succinate may enter the citric acid cycle, thus comple- ting the catabolic pathway of CH 3 -4-aminobutyrate generated from the pyrrolidine ring of nicotine. Results Expression of the pAO1 mao and sad-like genes required the presence of nicotine in the growth medium The mao and sad genes addressed in this study are located on pAO1 in a gene cluster flanked by a Tn554 element and an ORF of a truncated transposase (Fig. 2, panel A, DTn) [6]. This gene cluster contains the purU-mabO-folD operon, which is transcribed only in the presence of nicotine under the control of the transcriptional activator PmfR [9]. If the mao and sad- like genes were functionally connected to mabO, one Fig. 1. Formation and breakdown of c-N-methylaminobutyrate in A. nicotinovorans pAO1. C B. Chiribau et al. c-N-methylaminobutyrate catabolism FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS 1529 would expect them also to be expressed in a nicotine- dependent manner. In order to investigate this, we analyzed the transcription of these genes in the pres- ence and absence of nicotine in the growth medium by RT-PCR. The results presented in Fig. 2B confirmed the expectation that these genes are transcribed only in the presence of nicotine, as was the case for the mabO gene. The mao-like gene encodes an AO The mao gene expressed from pH6EX3 produced a fusion protein with an N-terminal extension reading MSPIHHHHHHLVPRGS V. The first amino acid of mao is valine (underlined V in the one letter amino acid code). The protein eluted from the nickel-chelat- ing sepharose column had an intense yellow colour, indicating formation of a flavoprotein. It showed a characteristic flavin spectrum with maxima at 450 nm and a shoulder at 470 nm (Fig. 3B). Examined by PAGE on 10% SDS gels, AO migrated in good accordance with its calculated relative molecular mass of 46 100 (Fig. 3A). When precipitated with 10% trichloroacetic acid (TCA), the sample formed a white protein pellet and a yellow supernatant, showing that the flavin cofactor was not covalently bound to the protein and thin layer chromatography (TLC) indicated that the cofactor was FAD (not shown). Gel permeation chromatography revealed that the protein was a monomer in solution (not shown). Monoamine oxidase activity could be also detected with 4-aminobutyrate as substrate, but surprisingly, the enzyme utilized CH 3 -4-aminobutyrate with high efficiency. It removed the secondary amine of CH 3 -4- aminobutyrate and the reaction products were methyl- amine (Fig. 3C) and succinic semialdehyde (see below). Thus, the enzyme behaved as an amine oxidase rather than as a monoamine oxidase. The pH optimum was found to be 9.8. The K m and k cat of AO with CH 3 -4- aminobutyrate as substrate was 2.5 ± 0.2 mm and 1230 ± 20 s )1 , respectively (Table 1), as compared with the previously determined K m of 140 lm and k cat of 800 s )1 for MABO [8]. It may be observed that the catalytic efficiency of MABO for CH 3 -4-aminobutyrate (k cat ⁄ K m of 5.71 lm )1 Æs )1 ) was approximately 10-fold higher as compared with that of AO (k cat ⁄ K m of 0.49 lm )1 Æs )1 ). With 4-aminobutyrate as substrate, the AO activity was much reduced (see Table 1). AO was inactive with the following compounds tested as substrates: spermidine, spermine, sarcosine, dimethylglycine, glycine, choline, betaine, a-methyla mino isobutyric acid, methylamine propionnitrile, methyl- amino propylamine. A B Fig. 2. pAO1 genes addressed in this study and RT-PCR analysis of transcripts. (A) Schematic representation of the pAO1 gene and ORF cluster flanked by Tn554 and DTn. The cluster consists of the pmfR gene, encoding the regulator of the purU-mabO-folD operon, a per- mease-like ORF, the genes of the purU-mabO-folD operon, two ORFs A and B resembling a multidrug efflux pump (MDR), the sad and mao genes of a succinate semialdehyde dehydrogenase and a monoamine oxidase, respectively, and ORF204 with unknown function. Arrows indicate the position of primers employed in the PCR amplification of gene fragments and the numbers the size in basepair of the amplified DNA fragment. (B) RT-PCR of RNA derived from A. nicotinovorans pAO1 grown in the presence (lanes 1–6) or absence (lanes 7–12) of nicotine in the growth medium. PCR was performed with RNA as negative control and cDNA as template, respectively, in the presence of primer pairs specific for mao (lanes 1, 2 and 7, 8), specific for sad (lanes 3, 4 and 9, 10) and specific for mabO (lanes 5, 6 and 11, 12). M, DNA size marker. c-N-methylaminobutyrate catabolism C B. Chiribau et al. 1530 FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS The pAO1 sad gene encodes a succinic semialdehyde dehydrogenase (SsaDH) The N-terminal extension of the recombinant SsaDH reads MSPIHHHHHHLVPRGS M (the start methio- nine residue is underlined). Analyzed by PAGE on 10% SDS gels, it migrated in good accordance with its calculated molecular mass of 51 kDa (Fig. 3A) and the native enzyme is a homodimer (not shown). The kinetic constants of the enzyme are listed in Table 1. When NAD + replaced NADP + in the assay, the activ- ity of the enzyme was about 25-fold less then that observed with NADP + . The reaction at 10 mm NAD + still did not reach saturation level. The enzyme was active also towards butyraldehyde (8.5% of the activity observed with succinic semialde- hyde) and propionaldehyde (1.6% of the activity observed with succinic semialdehyde) as substrates. Coupled assay with AO and SsaDH with CH 3 -4-aminobutyrate as substrate In order to confirm the formation of succinic semialde- hyde in the reaction of AO with CH 3 -4-aminobutyrate, a coupled assay was performed with AO and SsaDH. The SsaDH reaction was followed spectrophoto- metrically at 340 nm by the reduction of NADP + (Fig. 4A,B). The same SsaDH activity was determined in the coupled assay as the SsaDH activity determined with succinic semialdehyde as substrate. This identified this compound as the second product of the AO reac- tion with CH 3 -4-aminobutyrate. As expected, the reduction of NADP + was decreased when 4-aminobu- tyrate was employed as substrate in the coupled assay, demonstrating that AO deaminates 4-aminobutyrate to succinic semialdehyde with reduced efficiency. When AO was replaced with MABO in the coupled assay with CH 3 -4-aminobutyrate as substrate, no NADP + reduction was observed (Fig. 4A). This result was pre- dicted, as the product of the MABO reaction is 4-ami- nobutyrate, which is not a substrate for SsaDH. When, besides AO and SsaDH, increasing amounts of MABO were introduced in the coupled reaction with CH 3 -4-aminobutyrate as substrate, the measured NADPH production slowed down (Fig. 4B). This indi- cated that the two enzymes indeed competed for the same substrate. As MABO has an approximately 10-fold higher catalytic activity than AO, in its presence, the predominant reaction product is 4-aminobutyrate, and thus reduction of NADP + was slowed down. Since 4-aminobutyrate is also a poor substrate for AO, which in this case acts as a monoamine oxidase and transforms 4-aminobutyrate into succinic semialde- Table 1. Kinetic constants of enzymes described in this study. Enzyme Substrate K m (mM) k cat (s )1 ) k cat ⁄ K m (lM )1 Æs )1 ) AO c-N-methylaminobutyrate 0.25 ± 0.2 1230 ± 20 5.71 AO c-aminobutyrate 6.66 ± 0.16 878 ± 32 0.131 SsaDH Succinic semialdehyde 0.34 ± 0.1 23000 ± 700 67.6 SsaDH NADP + 0.13 ± 0.01 25000 ± 800 191 C B Absorbance (A) Wavelength (nm) AO SaD A 36 – 45 – 55 – 66 – 84 – kDa PA MAs EA MG MAp Origin 0.2 0.3 0.4 320 380 440 500 Fig. 3. Characterization of enzymes and identification by TLC of methylamine as reaction product of AO with CH 3 -4-aminobutyrate. (A) Analysis of purified proteins on 10% SDS gel. (B) UV-visible spectrum of the FAD-containing AO. (C) The AO reaction and TLC were performed as described in Experimental procedures. Four microliters of a 10 m M solution of propylamine (PA), methylamine (MAs) and ethylamine (EA) was applied as standard to the TLC. MG, CH 3 -4-aminobutyrate, which does not react with ninhydrin; MAp, methylamine formed in 5 lL of the AO reaction with CH 3 -4-aminobutyrate as substrate. C B. Chiribau et al. c-N-methylaminobutyrate catabolism FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS 1531 hyde, a certain level of SsaDH activity will be present, even at high MABO concentrations. [ 14 C]-labelled metabolites identified by TLC in the culture medium of A. nicotinovorans pAO1 grown in the presence of [ 14 C]nicotine The time-dependent analysis of [ 14 C]-labelled metabo- lites secreted by the bacteria into the growth medium revealed the situation shown in Fig. 5(A). Growth resumed with nicotine as carbon and nitrogen source and the cultures turned blue, an indication that nicotine breakdown was completed. In both situations, either with or without ammonium salts, labelled meth- ylamine was the predominant metabolite detected. Growth of A. nicotinovorans carrying or lacking pAO1 on minimal medium with CH 3 -4-aminobutyrate, 4-aminobutyrate or methylamine as carbon source Both A. nicotinovorans strains, either with or without plasmid pAO1, were able to grow on mineral salt med- ium with 4-aminobutyrate, but not with CH 3 -4-amino- butyrate or methylamine as carbon source (Fig. 5B). A B A Fig. 5. [ 14 C]Nicotine metabolites in the medium of A. nicotinovo- rans pAO1 and growth of A. nicotinovorans pAO1 and A. nicotino- vorans lacking pAO1 on CH 3 -4-aminobutyrate, 4-aminobutyrate and CH 3 NH 2 as carbon source. (A) Seven microliters of medium of a 10 mL culture grown for 1 h (lanes 2 and 6), for 2 h (lanes 3 and 7), for 3 h (lanes 4 and 8), and for 4 h (lanes 5 and 9) on minimal medium supplemented with [ 14 C]nicotine in the presence (lanes 2–5) or absence (lanes 6–9) of (NH 4 ) 2 SO 4 were analyzed on a TLC plate (see Experimental procedures). The plate was exposed for 62 h to an X-ray film. MA, position of methylamine standard stained with the ninhydrin reaction on the same plate; N, nicotine; X, unidentified labelled metabolite; Origin, site of application of sam- ples. (B) Arthrobacter strains were grown on minimal medium with the indicated carbon sources as described in Experimental procedures. n, A. nicotinovorans pAO1 and m, A. nicotinovorans lacking pAO1, grown on 4-aminobutyrate; X, A. nicotinovorans pAO1 and A. nicotinovorans lacking pAO1 grown on CH 3 -4-amino- butyrate or CH 3 NH 2 . B A Fig. 4. AO and SsaDH-coupled enzyme assay. (A) The NADPH pro- duction in the assay was determined with the additions as indicat- ed. The presence of AO, SsaDH and CH 3 -4-aminobutyrate as substrate were required for maximal activity. In the absence of AO there was no NADPH produced and with AO, SsaDH and 4-amino- butyrate as substrate the NADPH production was strongly reduced. (B) MABO and AO compete for CH 3 -4-aminobutyrate in vitro. NADPH production at constant 10 lg AO and 3 lg SsaDH decreas- es with increasing MABO concentrations in the coupled assay. c-N-methylaminobutyrate catabolism C B. Chiribau et al. 1532 FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS Discussion The MAO-like protein encoded by the mao gene of pAO1 was shown here to be an amine oxidase. Like polyamine oxidases [10–12] it acts upon a secondary amine, in this case CH 3 -4-aminobutyrate, giving rise to methylamine and succinic semialdehyde. Its activity was specific towards CH 3 -4-aminobutyrate and its monoamine oxidase activity with 4-aminobutyrate as substrate was weak. Similar to other members of the polyamine oxidases, the FAD cofactor was noncova- lently bound to the apoprotein and the C-terminal fingerprint sequence SGGCY of monoamine oxidases, with C being the cysteine residue to which the FAD cofactor is covalently attached in these enzymes [13], was replaced by the sequence AGGA 359 Y. The second enzyme characterized in this study showed high similarity to NADP + -dependent SsaDH from various organisms (not shown). It contains the amino acid consensus patterns of the aldehyde dehy- drogenases glutamic acid active site (SwissProt Prosite PS00687) in the form of ME 270 LGGNA, and cysteine acive site (SwissProt Prosite PS00070) in the form of GEAC 304 TAAN. The unexpected finding that CH 3 -4-aminobutyrate and not 4-aminobutyrate was the substrate of the AO and thus both MABO and AO have the same substrate led us to postulate two pathways for the catabolism of CH 3 -4-aminobutyrate that is generated from the side chain of 2,6-dihydroxypseudooxynicotine [7]. The first would start with the oxidative demethylation of CH 3 - 4-aminobutyrate by MABO and result in 4-aminobuty- rate, CH 2 TH 4 and reduced FADH 2 [8]. The methylene group of CH 2 TH 4 can be further oxidized by the gene products of folD and purU to formaldehyde. In the CH 2 TH 4 dehydrogenase ⁄ cyclohydrolase reaction, energy is conserved in NADPH and formaldehyde may be assimilated by the Embden–Meyerhof fructose- bisphosphate aldose ⁄ transaldolase variant of the ribu- lose monophosphate cycle [14,15]. The amino group of 4-aminobutyrate, the second reaction product in this pathway, may be transaminated to a-ketoglutarate and the remaining succinic semialdehyde may be oxidized to succinate by a succinic semialdehyde dehydrogenase [16,17]. This pathway for 4-aminobutyrate catabolism is generally found in bacteria [18–20]. It also appears to be active in A. nicotinovorans, independent of the presence of the megaplasmid pAO1, since both strains, with and without pAO1, were able to grow on 4-ami- nobutyrate as the carbon source. The second, pAO1-encoded pathway would start with the newly discovered reaction of CH 3 -4-aminobutyrate deamination to succinic semialdehyde and methylamine catalyzed by AO. In this reaction FAD is reduced to FADH 2 . The pAO1-encoded SsaDH then produces suc- cinate, which enters the citric acid cycle, and NADPH. A. nicotinovorans devoid of pAO1 was not able to grow on CH 3 -4-aminobutyrate. A. nicotinovorans pAO1 was able to grow on CH 3 -4-aminobutyrate only in the pres- ence of low amounts of nicotine added as inducer of the nicotine degradation pathway (Ganas and Brandsch, unpublished). Therefore, it is reasonable to assume that pAO1 encoded AO and SsaDH have evolved specifically for the catabolism of CH 3 -4-aminobutyrate produced from nicotine. Methylamine can be used by the facultative methylotroph Arthrobacter strain P1 [15], but A. nicotinovorans could not grow on methylamine, which instead appeared in the growth medium when the bacteria was grown in the presence of nicotine. Both pathways may lead to the complete mineraliza- tion of the pyrrolidine ring of nicotine, which after oxidation by 6-hydroxy-l-nicotine oxidase, is cleaved off from the pyridine ring of nicotine in the form of CH 3 -4-aminobutyrate. Each of these pathways starts with an enzyme specific for an unusual substrate. MABO may have derived from a sarcosine oxidase [8] by increasing its substrate specificity to CH 3 -4-amino- butyrate, a compound with two additional C-units as compared to sarcosine. AO still has a very low mono- amine oxidase catalytic activity towards 4-aminobuty- rate, but is specific for the oxidative deamination of the secondary amine of CH 3 -4-aminobutyrate. Appa- rently there was a selective pressure during the esta- blishment of nicotine catabolism for the evolution of new enzyme specificities starting from enzymes with sarcosine oxidase and polyamine oxidase activities. We must ask our selves which pathway predominates in vivo. From the in vitro kinetic data one would predict a preferentially channelling of CH 3 -4-aminobutyrate to the demethylation pathway, since the k cat ⁄ K m of MABO show it to be approximately 10 times more cata- lytically active than the deaminating AO. We do not know at the moment how the in vivo competition of the two enzymes for the same substrate is regulated. Addi- tional work will be required to answer this question. However, under the experimental conditions used, methylamine is secreted into the growth medium, which shows that the deamination pathway is active in vivo. Experimental procedures Bacterial strains and growth conditions A. nicotinovorans and A. nicotinovorans pAO1 were grown at 30 °C in citrate medium [21]. Alternatively, the citrate C B. Chiribau et al. c-N-methylaminobutyrate catabolism FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS 1533 was replaced, as indicated with CH 3 -4-aminobutyrate, 4-aminobutyrate or methylamine. Escherichia coli XL-Blue was employed both as host for plasmids and as expression strain and was grown in LB medium supplemented with the appropriate antibiotics at 37 °C. Chemicals and biochemicals Endonuclease restriction enzymes were purchased from New England Biolabs (Frankfurt, Germany), Pfu-Ultra DNA-polymerase and T4 reverse transcriptase from Strata- gene (Amsterdam, the Netherlands), Rapid DNA Ligation Kit from Roche Applied Science (Mannheim, Germany), nickel-chelating sepharose from Amersham Biosciences (Freiburg, Germany). [ 14 C]Nicotine (1.25 mCiÆ mmol )1 ), labelled at the methyl group was a kind gift of K. Decker (Freiburg, Germany). All other chemicals were obtained from Sigma (Steinheim, Germany) unless otherwise indica- ted and were of highest purity available. RT-PCR Total RNA was isolated from A. nicotinovorans cultures grown in the presence or absence of nicotine with the help of the RNeasy kit (Qiagen, Hilden, Germany), reverse-transcribed with T4 reverse transcriptase, and the respective cDNAs were applied as templates in PCR reac- tions as described previously [8,22] with primers listed in Table 2. Cloning of the monoamine oxidase (mao) and the succinate semialdehyde dehydrogenase (sad)-like genes The pAO1 DNA carrying the corresponding ORFs was amplified with the primer pair #1 and #2 for mao and #3 and #4 for sad (see Table 2), using Pfu-Ultra DNA-Poly- merase and pAO1 as template. The PCR conditions were 95 °C for 1 min, 54 °C for 45 s, 72 °C for 2 min, repeated 30 times and followed by 72 °C for 10 min. The amplified DNA and the vector pH 6EX3 [23] were digested with endonucleases BamHI and XhoI, ligated with the rapid DNA ligation kit (Roche Applied Sciences, Mannheim, Germany) and transformed into E. coli XL1-Blue compe- tent bacteria. Expression and purification of the recombinant proteins A 100 mL preculture of E. coli XL-1Blue harbouring pH 6EX3mao or pH 6EX3sad was diluted 1 : 10 in 1 L of LB medium. After 2 h at 37 °C, expression of the genes was induced for 4–5 h at 30 °C with 1 mm IPTG. Prepar- ation of bacterial extracts and purification of the proteins on High Performance nickel-chelating sepharose was as des- cribed previously [8]. The recombinant proteins were stable for several weeks at 4 °C with minor precipitation. The isolated proteins were analyzed by SDS ⁄ PAGE on 10% polyacrylamide gels. Superdex S-200 permeation chroma- tography, for determining the size of the native proteins, was performed with the aid of an A ¨ KTA device (Amer- sham Biosciences, Freiburg, Germany). Determination of enzyme activities AO activity was tested using the peroxidase coupled assay [8]. The 1-mL assay consisted of 20 mm potassium phos- phate buffer, pH 9.8, 0.0007% o-dianisidine, 10 U horse- radish peroxidase (Sigma), and 10 lg AO. The reaction was initiated by the addition of 10 mm substrate (CH 3 -4-ami- nobutyrate or 4-aminobutyrate). The oxidation of o-dianisi- dine was monitored at room temperature by the increase in absorption at 430 nm. SsaDH activity was measured in a 1 mL assay which contained: 100 mm sodium pyrophosphate buffer, pH 9, 5mm EDTA, 500 lm NAD + or NADP + and 1.5 lg SsaDH. The reaction was started by the addition of 1.5 mm succinic semialdehyde substrate. The reduction of NAD + or NADP + was monitored by the increase in absorption at 340 nm for 5 min at room temperature. Table 2. Oligonucleotides used in this study. No Sequence Use 15¢-GAG GTG GAT CCG TGG GCC GCA-3¢ Forward mao, cloning 25¢-GAA TGA CTC GAG CCG AAG TAA TC-3¢ Reverse mao, cloning 35¢-CTT CTG AGG ATC CCA AAT GAC AGT-3¢ Forward sad, cloning 45¢-CAT GTA AGC CCC CTC GAG TCG TTC AG-3¢ Reverse sad, cloning 55¢-CGT CAC GGT ATT CGA AGC C-3¢ Forward mao, RT-PCR 65¢-CAC TGG CTA ATT CCA GTG C-3¢ Reverse mao, RT-PCR 75¢-CAC TAG CGA AGA TGC CGT C-3¢ Forward sad, RT-PCR 85¢-CCA ACG CAG AAA CTC GGC-3¢ Reverse sad, RT-PCR 95¢-CGG CAT TAT CGG TGA CAG C-3¢ Forward mabO, RT-PCR 10 5¢-CGC GCA ACA CTG AGG GAC-3¢ Reverse mabO, RT-PCR c-N-methylaminobutyrate catabolism C B. Chiribau et al. 1534 FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS A coupled AO-SsaDH assay was performed in 1 mL con- sisting of: 100 mm sodium pyrophosphate buffer, pH 9, 5mm EDTA, 500 lm NADP + ,10lg AO (which retains 100% activity under these reaction conditions) and 1.5 lg SsaDH. The reaction was started by the addition of 10 mm CH 3 -4-aminobutyrate and the reduction of NADP + was monitored at 340 nm in an Ultrospec 3100 Spectrophoto- meter (Amersham Biosciences). TLC of the reaction products of the enzyme assays Identification of CH 3 NH 2 and 4-aminobutyrate produced in the enzyme assays with AO and MABO was performed by TLC on Polygram Cel300 plates (Macherey-Nagel, Du ¨ ren, Germany) with n-butanol ⁄ pyridine ⁄ acetic acid ⁄ H 2 O (10 : 15 : 3 : 12 v ⁄ v ⁄ v ⁄ v) as mobile phase [8]. The plates were developed by spraying with a 0.1% (v ⁄ v) ninhydrin solution in acetone. Identification of [ 14 C]methylamine in the medium of [ 14 C]nicotine grown A. nicotinovorans pAO1 A. nicotinovorans pAO1 bacteria grown to the stationary phase were harvested by centrifugation, washed twice with minimal salts medium and finally resuspended in minimal salts medium supplemented with l-[ 14 C]nicotine (200 lm)in the presence or absence of ammonium sulfate. Aliquots of the growth medium were removed at different time points and analyzed by TLC for the presence of [ 14 C]methylamine as described above. The TLC plates were exposed to Kodak X-Omat AR X-ray films (Sigma, Taufkirchen, Germany) for various times. Growth of A. nicotinovorans carrying or lacking pAO1 on CH 3 -4-aminobutyrate, 4-aminobutyrate or methylamine CH 3 -4-aminobutyrate, 4-aminobutyrate or methylamine (2 gÆL )1 ) replaced citrate as carbon source in the minimal medium [21] in these experiments. Biotin at 41 nm final con- centration was added as vitamin supplement to the bacterial cultures. An A. nicotinovorans overnight culture (150 lL) was diluted 100 times in sterile 50-mL Falcon tubes and growth was monitored by the increase in turbidity at 600 nm. Acknowledgements We thank I. Deuchler for excellent technical assistance, C. Brizio (University of Bari, Italy) for help with the kinetic data and C. Sandu (The Rockefeller University, New York, NY, USA) for critically reading the manu- script. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to RB. References 1 Hukkanen J, Jacob P & Benowitz NL (2005) Metabo- lism and disposition kinetics of nicotine. Pharmacol Rev 57, 79–115. 2 Brandsch R (2006) Microbiology and biochemistry of nicotine degradation. Appl Microbiol Biotechnol 69, 493–498. 3 Roduit JP, Wellig A & Kienert A (1997) Renewable functionalized pyridines derived from microbial metabo- lites of the alkaloid (S)-nicotine. 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