Tài liệu Báo cáo khoa học: Final steps in the catabolism of nicotine Deamination versus demethylation of c-N-methylaminobutyrate doc

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. Heterocycles 45, 1687–
1702.
4 Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts
M & Witholt B (2001) Industrial biocatalysis today and
tomorrow. Nature 409, 258–268.
5 Malphettes L, Weber CC, El-Baba MD, Schoenmakers
RG, Aubel D, Weber W & Fussenegger M (2005) A
novel mammalian expression system derived from com-
ponents coordinating nicotine degradation in Arthrobac-
ter nicotinovorans pAO1. Nucleic Acids Res 33, e107.
6 Igloi GL & Brandsch R (2003) Sequence of the 165-
kilobase catabolic plasmid pAO1 from Arthrobacter
nicotinovorans and identification of a pAO1-dependent
nicotine uptake system. J Bacteriol 185, 1976–1986.
7 Sachelaru P, Schiltz E, Igloi GL & Brandsch R (2005)
An a ⁄ b-fold C–C bond hydrolase is involved in a cen-
tral step of nicotine catabolism by Arthrobacter nicotino-
vorans. J Bacteriol 187, 8516–8519.
8 Chiribau C-B, Sandu C, Fraaije M, Schiltz E &
Brandsch R (2004) A novel c-N-methylaminobutyrate
demethylating oxidase involved in catabolism of the

tobacco alkaloid nicotine by Arthrobacter nicotinovorans
pAO1. Eur J Biochem 271, 4677–4684.
9 Chiribau C-B, Sandu C, Igloi GL & Brandsch R
(2005) Characterization of PmfR, the transcriptional
activator of the pAO1-borne purU-mabO-folD operon
of Arthrobacter nicotinovorans. J Bacteriol 187, 3062–
3070.
10 Landry J & Sternglanz R (2003) Yeast Fms1 is a
FAD-utilizing polyamine oxidase. Biochem Biophys Res
Commun 303, 771–776.
11 Busch K, Piehler J & Fraomm H (2000) Plant succinic
semialdehyde dehydrogenase: dissection of nucleotide
binding by surface plasmon resonance and fluorescence
spectroscopy. Biochemistry 39, 10110–10117.
12 Tavladorakiu P, Schinina ME, Cecconi F, Di Agostino
S, Manera F, Rea G, Mariottini P, Federico R &
Angelici R (1998) Maize polyamine oxidase: primary
structure from protein and cDNA sequencing. FEBS
Lett 426, 62–66.
13 De Colibus L, Li M, Binda C, Lustig A, Edmondson
DE & Mattevi A (2005) Three-dimensional structure of
human monoamine oxidase A (MAO A): relation to the
structures of rat MAOA and human MAOB. Proc Natl
Acad Sci USA 102, 12684–12689.
C B. Chiribau et al. c-N-methylaminobutyrate catabolism
FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS 1535
14 Levering PR, Binnema DJ, van Dijken JP & Harder W
(1981) Enzymatic evidence for a simultaneous operation
of two one-carbon assimilation pathways during growth
of Arthrobacter P1 on choline. FEMS Lett 12, 19–25.

15 Xiaping Z, Fuller JH & McIntire W (1993) Cloning,
sequencing, expression, and regulation of the structural
gene for the Copper ⁄ Topa quinone-containing methyl-
amine oxidase from Arthrobacter strain P1, a Gram-
positive facultative methylotroph. J Bacteriol 175,
5617–5627.
16 Bartsch K, von Johnn-Marteville A & Schulz A (1990)
Molecular analysis of two genes of the Escherichia coli
gab cluster: nucleotide sequence of the glutamate: succi-
nic semialdehyde transaminase gene (gabT) and charac-
terization of the succinic semialdehyde dehydrogenase
gene (gabD). J Bacteriol 172, 7035–7042.
17 Chambliss KL, Caudle DL, Hinson DD, Moomaw CR,
Slaugther CA, Jakobs C & Gibson M (1995) Molecular
cloning of the mature NAD+-dependent succinic semi-
aldehyde dehydrogenase from rat and human. J Biol
Chem 270, 461–467.
18 Dover S & Halpern YS (1972) Utilization of c-amino-
butyric acid as the sole carbon and nitrogen source by
Escherichia coli K-12 mutants. J Bacteriol 109, 835–843.
19 Metzer E & Halpern YS (1990) In vivo cloning and
characterization of the gabCTDP gene cluster of Escher-
ichia coli K-12. J Bacteriol 172, 3250–3256.
20 Niegemann E, Schulz A & Bartsch K (1993) Molecular
organization of the Escherichia coli gab cluster: nucleo-
tide sequence of the structural genes gabD and gabP
and expression of the 4-aminobutyrate permease gene.
Arch Microbiol 160, 454–460.
21 Bru
¨

hmu
¨
ller M, Schimz A, Messmer L & Decker K
(1975) Covalently bound FAD in d-6-hydroxynicotine
oxidase. J Biol Chem 250, 7747–7751.
22 Sandu C, Chiribau C-B & Brandsch R (2003) Charac-
terization of HdnoR, the transcriptional repressor of the
6-hydroxy- d -nicotine oxidase gene of Arthrobacter
nicotinovorans pAO1, and its DNA-binding activity in
response to l- and d-nicotine derivatives. J Biol Chem
278, 51307–51315.
23 Berthold H, Scanarini M, Abney CC, Frorath B &
Northemann W (1992) Purification of recombinant anti-
genic epitopes of the human 68-kDa (U1) ribonucleo-
protein antigen using the expression system pH6EX3
followed by metal chelating affinity chromatography.
Protein Expr Purif 3, 50–56.
c-N-methylaminobutyrate catabolism C B. Chiribau et al.
1536 FEBS Journal 273 (2006) 1528–1536 ª 2006 The Authors Journal compilation ª 2006 FEBS