Several soil microorganisms have evolved the enzymatic ability to mineralize nicotine, Keywords amine oxidase; Arthrobacter nicotinovorans; nicotine; c-N-methylaminobutyrate; succinic se
Trang 1Deamination 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-aminobuty-4-aminobuty-rate 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 Kmof 2.5 mm and kcatof 1230 s)1for amine oxidase vs Kmof
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; 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.
Trang 2but only the enzymes of nicotine catabolism of
Arthrobacter nicotinovoranspAO1 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 (CH3
-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 CH3 -4-aminobuty-rate It consists of mabO, encoding the enzyme c-N-methylaminobutyrate oxidase (MABO, Fig 1), which oxidatively demethylates CH3-4-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 (CH2TH4) 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 CH3-4-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 CH3-4-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.
Trang 3would 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 abspres-ence 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
MSPIHHHHHHLVPRGSV 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 CH3-4-aminobutyrate with high efficiency It removed the secondary amine of CH3 -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 Km and kcat of AO with CH3 -4-aminobutyrate as substrate was 2.5 ± 0.2 mm and
1230 ± 20 s)1, respectively (Table 1), as compared with the previously determined Km of 140 lm and kcat
of 800 s)1 for MABO [8] It may be observed that the catalytic efficiency of MABO for CH3-4-aminobutyrate (kcat⁄ Km of 5.71 lm)1Æs)1) was approximately 10-fold higher as compared with that of AO (kcat⁄ Km of 0.49 lm)1Æs)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-methylamino 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.
Trang 4The pAO1 sad gene encodes a succinic
semialdehyde dehydrogenase (SsaDH)
The N-terminal extension of the recombinant SsaDH
reads MSPIHHHHHHLVPRGSM (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
CH3-4-aminobutyrate as substrate
In order to confirm the formation of succinic semialde-hyde in the reaction of AO with CH3-4-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 CH3-4-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 CH3-4-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 CH3-4-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 Km(m M ) kcat(s)1) kcat⁄ K m (l M )1Æs)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
Wavelength (nm)
A
kDa
Origin
0.2 0.3 0.4
Fig 3 Characterization of enzymes and identification by TLC of
methylamine as reaction product of AO with CH3-4-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.
Trang 5hyde, a certain level of SsaDH activity will be present,
even at high MABO concentrations
[14C]-labelled metabolites identified by TLC in the
culture medium of A nicotinovorans pAO1
grown in the presence of [14C]nicotine
The time-dependent analysis of [14C]-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
CH3-4-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 CH3 -4-amino-butyrate or methylamine as carbon source (Fig 5B)
A
B
Fig 5 [ 14 C]Nicotine metabolites in the medium of A nicotinovo-rans pAO1 and growth of A nicotinovonicotinovo-rans pAO1 and A nicotino-vorans lacking pAO1 on CH3-4-aminobutyrate, 4-aminobutyrate and
CH3NH2as 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 CH3-4-aminobutyrate in vitro.
NADPH production at constant 10 lg AO and 3 lg SsaDH
decreas-es with increasing MABO concentrations in the coupled assay.
Trang 6The 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 CH3-4-aminobutyrate, giving rise to
methylamine and succinic semialdehyde Its activity
was specific towards CH3-4-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 AGGA359Y
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 ME270LGGNA, and cysteine
acive site (SwissProt Prosite PS00070) in the form of
GEAC304TAAN
The unexpected finding that CH3-4-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
CH3-4-aminobutyrate that is generated from the side
chain of 2,6-dihydroxypseudooxynicotine [7] The first
would start with the oxidative demethylation of CH3
-4-aminobutyrate by MABO and result in
4-aminobuty-rate, CH2TH4and reduced FADH2[8] The methylene
group of CH2TH4can be further oxidized by the gene
products of folD and purU to formaldehyde In
the CH2TH4 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 CH3-4-aminobutyrate
deamination to succinic semialdehyde and methylamine
catalyzed by AO In this reaction FAD is reduced to FADH2 The pAO1-encoded SsaDH then produces suc-cinate, which enters the citric acid cycle, and NADPH
A nicotinovoransdevoid of pAO1 was not able to grow
on CH3-4-aminobutyrate A nicotinovorans pAO1 was able to grow on CH3-4-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 CH3-4-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
CH3-4-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 CH3 -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 CH3-4-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 CH3-4-aminobutyrate
to the demethylation pathway, since the kcat⁄ Km 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 30C in citrate medium [21] Alternatively, the citrate
Trang 7was replaced, as indicated with CH3-4-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 37C
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) [14C]Nicotine (1.25 mCiÆmmol)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
95C for 1 min, 54 C for 45 s, 72 C for 2 min, repeated
30 times and followed by 72C 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 37C, expression of the genes was induced for 4–5 h at 30C with 1 mm IPTG Prepar-ation of bacterial extracts and purificPrepar-ation of the proteins
on High Performance nickel-chelating sepharose was as des-cribed previously [8] The recombinant proteins were stable for several weeks at 4C 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 (CH3 -4-ami-nobutyrate or 4-ami-4-ami-nobutyrate) 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,
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.
1 5¢-GAG GTG GAT CCG TGG GCC GCA-3¢ Forward mao, cloning
2 5¢-GAA TGA CTC GAG CCG AAG TAA TC-3¢ Reverse mao, cloning
3 5¢-CTT CTG AGG ATC CCA AAT GAC AGT-3¢ Forward sad, cloning
4 5¢-CAT GTA AGC CCC CTC GAG TCG TTC AG-3¢ Reverse sad, cloning
5 5¢-CGT CAC GGT ATT CGA AGC C-3¢ Forward mao, RT-PCR
6 5¢-CAC TGG CTA ATT CCA GTG C-3¢ Reverse mao, RT-PCR
7 5¢-CAC TAG CGA AGA TGC CGT C-3¢ Forward sad, RT-PCR
8 5¢-CCA ACG CAG AAA CTC GGC-3¢ Reverse sad, RT-PCR
9 5¢-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
Trang 8A coupled AO-SsaDH assay was performed in 1 mL
con-sisting of: 100 mm sodium pyrophosphate buffer, pH 9,
5 mm EDTA, 500 lm NADP+, 10 lg AO (which retains
100% activity under these reaction conditions) and 1.5 lg
SsaDH The reaction was started by the addition of 10 mm
CH3-4-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 CH3NH2 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 ⁄ H2O
(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 [14C]methylamine in the medium
of [14C]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-[14C]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 [14C]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 CH3-4-aminobutyrate, 4-aminobutyrate
or methylamine
CH3-4-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
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