Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange between the nortropine and norpseudotropine catabolic pathways

Article abstract of DOI:10.1016/j.phytol.2014.04.014

Selected strains of Pseudomonas bacteria can degrade tropane alkaloids to obtain both nitrogen and carbon for growth. In order to probe the mechanisms of the catabolic enzymes involved, the metabolic process responsible for the opening of the 8-azabicyclo[3.2.1]octan-3-ol ring of nortropane alkaloids has been explored. It is found that the bacteria contain considerable flexibility in their enzyme complement and can convert (3-endo)-8-azabicyclo[3.2.1]octan-3-ol) (nortropine (2) to (3-exo)-8-azabicyclo[3.2.1]octan-3-ol) (norpseudotropine). Both of these compounds can serve as substrates for the catalytic cascade. In order to establish the proportionation between direct and indirect pathways, metabolism has been probed by competitive substrate availability and by incorporation of stable heavy labels into substrate pools. The results indicate that, while norpseudotropine is almost entirely metabolized directly, nortropine is partitioned c. 4:1 between direct and indirect catabolism.

Full text of DOI:10.1016/j.phytol.2014.04.014

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PHYTOL 703 1–9
Phytochemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
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Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures:
Interchange between the nortropine and norpseudotropine catabolic
pathways
a
a
´
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6
Q1 Katarzyna Kosieradzka , Pauline Le Faouder , Roland Molinie a,b, Emmanuel Gentil a,
Jacques Lebreton b, Richard J. Robins a,
*
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a Elucidation of Biosynthesis by Isotopic Spectrometry Group, Unit for Interdisciplinary Chemistry: Synthesis-Analysis-Modelling (CEISAM),
`
University of Nantes-CNRS UMR6230, 2, rue de la Houssiniere, 44322 Nantes, France
b Multistep Synthesis and Bioscience Group, Unit for Interdisciplinary Chemistry: Synthesis-Analysis-Modelling (CEISAM),
10
University of Nantes-CNRS UMR6230, 2, rue de la Houssinie`re, 44322 Nantes, France
A R T I C L E I N F O
A B S T R A C T
Article history:
Selected strains of Pseudomonas bacteria can degrade tropane alkaloids to obtain both nitrogen and
carbon for growth. In order to probe the mechanisms of the catabolic enzymes involved, the metabolic
process responsible for the opening of the 8-azabicyclo[3.2.1]octan-3-ol ring of nortropane alkaloids has
been explored. It is found that the bacteria contain considerable flexibility in their enzyme complement
and can convert (3-endo)-8-azabicyclo[3.2.1]octan-3-ol) (nortropine (2) to (3-exo)-8-azabicy-
clo[3.2.1]octan-3-ol) (norpseudotropine). Both of these compounds can serve as substrates for the
catalytic cascade. In order to establish the proportionation between direct and indirect pathways,
metabolism has been probed by competitive substrate availability and by incorporation of stable heavy
labels into substrate pools. The results indicate that, while norpseudotropine is almost entirely
metabolized directly, nortropine is partitioned c. 4:1 between direct and indirect catabolism.
ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Received 30 January 2014
Received in revised form 15 April 2014
Accepted 25 April 2014
Available online xxx
Keywords:
Pseudomonas strains 1T3 and MS2
Nor(pseudo)tropine, Catabolism
Stable isotope label
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1. Introduction
ubiquitously active in xenobiotic metabolism (Meunier et al., 25
2004).
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The pharmaceutical value of the tropane alkaloid esters,
hyoscyamine (atropine), scopolamine and cocaine, has been
recognized for many years. These compounds, which accumulate
in the leaves of several members of the plant families Solanaceae
and Erythroxylaceae, are, however, highly toxic. While their
turnover in plants has been demonstrated to some extent
(Kitamura et al., 1992), the complete catabolism of neither the
tropane nor the acidic moiety has been characterized. In contrast,
their degradation in both liver and Pseudomonas bacteria has
received considerable attention. In the mammalian liver, cocaine
degradation has been shown (Kloss et al., 1983) to be one of the
many functions of the cytochrome P450 monooxygenases
Many members of the Pseudomonadaceae bacteria are recog- 27
nized for their ability to metabolize a wide range of compounds, 28
including alkaloids (Rathbone and Bruce, 2002; Rathbone et al., 29
2002), principally due to the extreme versatility of their 30
complement of cytochrome P450 mixed-function oxidases (Cryle 31
et al., 2003). A Pseudomonas sp. (strain AT3 and a mutant derived 32
therefrom, MS2) with the inducible ability to grow on atropine, 33
hyoscyamine or tropine as sole carbon and nitrogen source has 34
been isolated from the rhizosphere soil of the tropane-alkaloid- 35
producing plant Atropa belladonna (Bartholomew et al., 1996; Long 36
et al., 1993). In this isolate, degradation of the tropine (1, (3-endo)- 37
8-methyl-8-azabicyclo[3.2.1]octan-3-ol) moiety involves N-de- 38
methylation as the first step in the excision of the nitrogen from 39
the ring (Fig. 1). The product of N-demethylation, nortropine (2, (3- 40
endo)-8-azabicyclo[3.2.1]octan-3-ol), undergoes scission of one C– 41
N bond in the azabicyclo[3.2.1] structure, hypothetically yielding 42
the cyclic amine 3, which by deamination yields 6-hydroxycyclo- 43
hepta-1,4-dione (4), a compound shown to accumulate in the 44
dehydrogenase-deficient strain Pseudomonas MS2 (Bartholomew 45
et al., 1995). In strain AT3, dehydrogenase activity reduces 4 to 46
*
Corresponding author. Tel.: +33 251125701; fax: +33 251125712.
Q2
E-mail addresses: kkosieradzka@interia.pl (K. Kosieradzka),
pauline.le-faouder@inserm.fr (P. Le Faouder),
roland.molinie@u-picardie.fr (R. Molinie´),
Emmanuel.Gentil@univ-nantes.fr (E. Gentil),
Jacques.Lebreton@univ-nantes.fr (J. Lebreton),
richard.robins@univ-nantes.fr (R.J. Robins).
http://dx.doi.org/10.1016/j.phytol.2014.04.014
1874-3900/ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014

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CH3
H
NH2
O
N
5
N
1
(iii)
(ii)
(i)
Degradation
products
OH
OH
3
O
O
HO
HO
Tropine
(1)
6-hydroxycyclohepta-1,4-dione
Nortropine
(3)
(4)
(2)
Fig. 1. General scheme for the degradation of tropine by Pseudomonas AT3.
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cyclohepta-1,3,5-trione (Bartholomew et al., 1993), which under-
goes cleavage to succinate and acetone.
nortropine present and to eliminate any isotope effect due to the
conversion to norpseudotropine.
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These Pseudomonas can also exploit tropinone (8-methyl-8-
azabicyclo[3.2.1]octan-3-one) and pseudotropine ((3-exo)-8-
methyl-8-azabicyclo[3.2.1]octan-3-ol) in the same manner.
The ability to interconvert tropine and tropinone was shown
to be due to the action of a well-characterized inducible NADP+-
dependent oxidoreductase (Bartholomew et al., 1995) but, in
contrast, no interconvertion of pseudotropine and tropinone
was observed. This was substantiated by a lack of appropriate
oxidoreductase activity in both crude extract and purified
protein. This is in contrast to the situation in planta, where two
distinct tropinone reductases (TR) exist, converting tropinone
exclusively to tropine (TR1) or pseudotropine (TR2) (Dra¨ger,
2006). It was further shown that nortropine could serve as
substrate for growth, that traces of nortropine were found in the
Subsequently, although previous reports concluded that the
metabolism of tropinone only yields tropine in Pseudomonas
strains AT3 and MS2 (Bartholomew et al., 1995), we have
demonstrated by using in vivo 13C NMR that both tropine and
pseudotropine are produced (ratio c. 95:5) by Pseudomonas AT3
cultures grown on tropinone (Bartholomeusz et al., 2008). In view
of these apparent discrepancies, we felt it necessary to re-evaluate
the interconversions between the two pathways. This has been
carried out using a series of approaches, including substrate
competition, isotope retention, isotope flux, and enzyme activities
with the aim of defining the metabolic route to the norpseudo-
tropine observed.
In this paper we report a series of studies carried out in order
to describe in greater detail the metabolic relationship between
nortropine, nortropinone and norpseudotropine with two
principal objectives. First, we needed to ascertain whether
norpseudotropine is directly degraded in the cultures. Second ,
growth medium, and that nortropine was oxidized at
a
comparable rate to tropine by the purified oxidoreductase,
while norpseudotropine was not a substrate (Bartholomew
et al., 1995). Based on these data and that no other oxidoreduc-
tase metabolizing these substrates could be found, the authors
reasonably concluded that there are two separate pathways, one
for tropine and tropinone via nortropine, the other for
pseudotropine via norpseudotropine. This conclusion was
reinforced by their demonstrating that in vivo 2H-label from
[3-2H]pseudotropine was retained at c. 90% in 6-hydroxycyclo-
hepta-1,4-dione (4) but only at c. 70% for [3-2H]tropine
(Bartholomew et al., 1995). This supported the concept that
only the latter alkaloid undergoes significant conversion to
tropinone and that, in view of the lack of any activity to convert
tropinone to pseudotropine, the tropinone thus produced is
entirely reconverted to tropine.
we wanted to carry out
a preliminary evaluation of the
similarity of the TR1- and TR2-type oxidoreductases in
Pseudomonas to the well-described plant enzymes, for which
tropinone is the natural substrate.
2. Results and discussion
124
2.1. Metabolism of nortropine, norpseudotropine and nortropinone by
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Pseudomonas AT3
Preliminary studies on the metabolism of nortropine by the
Pseudomonas AT3, showed a transient accumulation of norp-
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´
seudotropine in the medium (Mayant, 2005; Molinie, 2005). To
Thus Pseudomonas AT3 and MS2 appeared to be an ideal model
clarify the link between metabolism of these two alkaloids,
growth curves and alkaloid profiles were established on
nortropine alone, norpseudotropine alone and a mixture of
the two (Fig. 2).
From Fig. 2a and c, it can be deduced that growth on these two
substrates was comparable. From the data presented in Fig. 2b, it
can be seen that as nortropine was consumed, a transient
accumulation of norpseudotropine and, to a lesser extent, of
nortropinone occurred. At 11 h, the concentrations of nortropine
and norpseudotropine were equivalent (c. 1.2 mM). By 12 h, all
substrate was consumed. These results clearly show that there is
an active process of transformation of nortropine into norpseudo-
tropine via nortropinone.
system in which to study the mechanism of tropine N-demethyla-
15
´
tion, probed through the associated N isotope effects (Molinie,
´
2005; Molinie et al., 2007; Robins et al., 2007). To determine the
isotope effects, small changes in the ratio of heavy/light isotope
need to be measured, either on the residual substrate or on the
product accumulated after a defined level of advancement of the
reaction. These Pseudomonas strains are an ideal model system to
study isotope effects since (i) tropine quantitatively sustains
growth, (ii) it is rapidly degraded, (iii) no (AT3) or only trace (MS2)
amounts of tropinone, pseudotropine or nortropine are found at
´
any time in the growth period (Molinie, 2005). Thus, the residual
substrate is readily isolated and the isotope ratio obtained
´
(Molinie, 2005).
When norpseudotropine is exhibited as substrate, however, a
different picture emerges. As shown in Fig. 2d, only traces of
nortropine and nortropinone were observed. This might indicate
that the interconvertion is mono-directional:
However, on turning our attention to the second step – the
opening of the nortropine ring (Fig. 1, step (ii)) – the transient
formation in nortropine-grown cultures of significant quantities
of norpseudotropine was observed in preliminary studies
(Mayant, 2005). This was in contrast to the previous report
(Bartholomew et al., 1996) and indicated that, at the level of the
nor-alkaloids, inter-pathway exchange could occur. If this were
the case, it would be difficult to define the concentration of
14476
nortropine ! nortropinone ! norpseudotropine
However, it cannot be ruled out that norpseudotropine is
converted to nortropine and that this latter is much more rapidly
14
5
89
0
151
Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014

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3
Fig. 2. Growth and metabolic characteristics of the Pseudomonas AT3 cultures. (a) and (b) growth and metabolite concentrations for growth on nortropine (7.7 mM initial
concentration); (c) and (d) growth and metabolite concentrations for growth on norpseudotropine (7.8 mM initial concentration); (a) and (b) growth and metabolite
concentrations for growth on a mixed substrate of nortropine and norpseudotropine (1.73 and 2.83 mM initial concentrations, respectively).
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metabolized, hence it does not accumulate. This second option
could be eliminated, however, from the observed metabolism of
nortropinone. A distinct lag period was seen, after which growth
was rapid (Fig. 3a) and both nortropine and norpseudotropine were
found in the medium (Fig. 3b), the former at about 2.5-fold the
concentration of the latter. This indicated unequivocally that 157
nortropinonecanbeconvertedtonortropine, aswefoundpreviously 158
for tropinone conversion to tropine (Bartholomeusz et al., 2008):
116509
nortropine nortropinone ! norpseudotropine
Fig. 3. Growth and metabolic characteristics of the Pseudomonas AT3 cultures. (a) and (b) growth and metabolite concentrations for growth on nortropinone (7.8 mM initial
concentration).
Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014

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K. Kosieradzka et al. / Phytochemistry Letters xxx (2014) xxx–xxx
Table 1
(TR) activities. In plants of the Solanaceae, two distinct enzymes
have been characterized, TR1 and TR2, which convert (nor)tropi-
none to (nor)tropine or (nor)pseudotropine, respectively. In a prior
study of activities in crude extracts made from Pseudomonas AT3, a
TR activity oxidizing both tropine and nortropine (c. 1.5 times
higher for nortropine) was identified. In contrast, no activity with
pseudotropine and norpseudotropine was found (Bartholomew
et al., 1995). Furthermore, when these authors presented
tropinone to the extract, only tropine was obtained.
In view of our in vivo evidence presented above that nortropine
can be converted to norpseudotropine, we investigated in greater
detail the tropinone oxidoreductase activities, notably in relation to
their activity with the nor-compounds (Table 1). We also tested the
known inhibitors of plant TR, 8-thiabicyclo[3.2.1]octane-3-one
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Specific activity of enzymes for tropane alkaloid metabolism in crude extracts of
Pseudomonas AT3.
Substrate
Activitya (pkat/mg protein)
Inhibitor
None
TBON
b-TBOL
Tropine
166
4
149
5
188
4
Pseudotropine
Tropinone
643
263
119
1293
0
630
209
101
1157
–
556
240
94
Nortropine
Norpseudotropine
Nortropinone
TBON
1118
–
b-TBOL
0
–
–
a
Each measurement of activity was repeated 5 times.
(TBON) and
b-8-thiabicyclo[3.2.1]octane-3-ol (b-TBOL) (Dra¨ger,
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6
12
3
The inter-relationship was further probed by growing cultures on
2006; Parr et al., 1991), foraction against the Pseudomonas activities.
From the data presented in Table 1, it can be deduced that under the
analytical conditions used, the in vitro activity is: (i) 42-fold higher
for tropine than for pseudotropine but only 2.2-fold higher for
nortropine than for norpseudotropine; (ii) 2.0-fold higher for
nortropinone than for tropinone; (iii) significantly higher in the
direction of oxidation, 3.9- and 4.9-fold, respectively, for the methyl-
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a mixture of nortropine (1.73 mM) and norpseudotropine(2.83 mM)
as co-substrates (Fig. 2e and f). The growth kinetics were similar to
those observed on either one of the substrates alone. The metabolic
profile for the two substrates differed markedly, however. Over the
first 6 h of culture the concentration of nortropine constantly
decreases to about 10% initial value, while the norpseudotropine
remained at about 95% initial value. Only after the exhaustion of
nortropine was a decrease in the concentration observed.
Two hypotheses can be advanced to explain these observations.
Either nortropine is the preferred substrate and is consumed
preferentially, norpseudotropine being metabolized only after
depletion of nortropine, or both substrates are metabolized
simultaneously, but norpseudotropine utilization is offset by the
isomerization of nortropine.
and nor-compounds; (iv) neither TBON nor
b-TBOL inhibit the TR
activity detected in Pseudomonas AT3. Moreover, incubation with
tropinone produced onlytropine, asfoundpreviously(Bartholomew
et al., 1995). In this, however, there is a discrepancy between the in
vitro and in vivo data: incubation of cultures with tropinone
produced a 95:5 mixture of tropine and pseudotropine (Bartholo-
meusz et al., 2008). Furthermore, incubation in vitro with
nortropinone produced
a 75:25 mixture of nortropine and
norpseudotropine, more in agreement with the in vivo data.
While the observations for tropine and pseudotropine are
broadly consistent with the results of Bartholomew et al. (1995),
they differ in showing a low but measurable oxidoreductase activity
with pseudotropine. They also indicate that the substrateutilization
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2.2. Analysis of the tropinone oxidoreductase activities in vitro
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Interconvertion of nortropine and norpseudotropine via nor-
tropinone requires the intervention of tropinone oxidoreductase
Fig. 4. Metabolism of [2H-3]alkaloids by Pseudomonas MS2 (for clarity of interpretation, the numbering of the tropane ring is retained).
Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014

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for the nor-series is markedly different from that for the methyl-
series. Particularly notable is that norpseudotropine is a good
substrate, while pseudotropine is a very poor substrate. They also
differ markedly from the substrate and inhibitor specificities of the
plant enzymes from the Solanaceae. In extracts from Datura
stramonium, tropinone was reduced to tropine with a 6-fold greater
rate than to pseudotropine (Portsteffen et al., 1992). In addition, TR I
reductive activity (tropinone!tropine) was stronger than the
oxidative activity (tropine!tropinone) (Portsteffen et al., 1994).
Moreover, the TR activity detected in Pseudomonas AT3 is insensitive
this experiment, also including as substrates [3-2H]nortropine and 271
[3-2H]norpseudotropine (Table 2).
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As can be seen from Table 2, the incorporations obtained from 273
[3-2H]tropine (68%) and [3-2H]pseudotropine (91%) as substrate 274
closely match those reported in the literature: 71% from 275
[3-2H]tropine and 93% for [3-2H]pseudotropine, respectively 276
(Bartholomew et al., 1995), confirming that a significant level of 277
to TBON and b-TBOL, both of which inhibited the TR2 purified from
D. stramonium (Dra¨ger et al., 1992). However, as recently reported,
reduction of tropane alkaloids is not exclusive to the short-chain
oxidoreductases described for plants of the Solanaceae but can also
be catalyzed by members of the aldo-keto reductase family
(Jirschitzka et al., 2012). It may be that the Pseudomonas AT3
enzymeisnot thereforeclosely related totheD. stramonium enzyme.
Taking together the observations made in vitro and in vivo, it can
be concluded that:
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3
6
7
(i) with tropine as substrate, pseudotropine formation is never
observed in vivo;
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0
(ii) with tropinone as substrate only tropine is found in vitro but
both tropine and pseudotropine are found in vivo;
(iii) with nortropine as substrate, norpseudotropine formation is
observed in vivo, indicating isomerization via nortropinone.
This is consistent with the results of in vitro studies on the
enzyme extracts, where nortropinone is reduced to nortropine
and norpseudotropine, and, indirectly with the in vivo
formation of pseudotropine from tropinone;
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3
24
5
9
0
(iv) the enzyme activity/activities present can interconvert
tropine tropinone!pseudotropine and nortropine nortro-
pinone!norpseudotropine but with very different level of
activity;
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5
4
5
(v) TR activity in vitro is higher in the direction of reduction, which
is consistent with observations made in vivo where no more
than traces of the ketone compounds are ever seen.
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2.3. Analysis of the interconvertion of nortropine and
norpseudotropine using isotopically enriched substrates
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According to the results presented above, it would appear that
epimerization at the level of nortropine$norpseudotropine occurs
at a much higher rate than at the level of tropine$pseudotropine.
Since epimerization will lead to the loss of the hydrogen from the 3
position, retention of 2H at the 3 position is indicative of direct
metabolism (Fig. 4). This approach was effectively adopted by
previous authors, who exploited their oxidoreductase-deficient
strain Pseudomonas MS2 to observe the incorporation of 2H from
[3-2H]tropine and [3-2H]pseudotropine into 6-hydroxycyclo-
hepta-1,4-dione (4), which accumulate in the medium (Bartholo-
mew et al., 1995). In order to obtain comparable data, we repeated
Fig. 5. Isotopic profile for growth and metabolism with nortropine and
norpseudotropine as co-substrates (1.73 and 2.83 mM initial concentrations,
respectively). (a) Measured values of d15N (%) from initial values of nortropine
Table 2
(
d15N = 25.6%) and norpseudotropine d15N = 0.8%). Complete growth and
Level of incorporation of 2H into 6-hydroxycyclohepta-1,4-dione by Pseudomonas
metabolite profiles are shown in Fig. 2e and f. (b) Concentrations of
norpseudotropine depending on the degree of conversion of nortropine to
norpseudotropine: NPSTRI mes= measured values, NPSTRI + 18%NTRI= calculated
values for 0% degradation of pseudotropine plus 18% nortropine converted to
norpseudotropine. (c)Predictedvaluesofd15N(%)ofnorpseudotropinedependingon
the degree of metabolism of norpseudotropine and of conversion of nortropine to
norpseudotropine. Legend: NPSTRI = experimental values for norpseudotropine (a);
18%N = calculated values for norpseudotropine with a contribution of 18% from
nortropine during the phase of rapid nortropine consumption (see Fig. 2f);
18%P = calculated values for norpseudotropine with a contribution of 18% from
nortropine during the phase of rapid norpseudotropine consumption (see Fig. 2f). See
Supplementary Information for model details.
MS2.
Substratea
Retention of 2H [%]
TBON (mM)
0
3.5
[3-2H]tropine
68
91
83
91
68
92
83
91
[3-2H]pseudotropine
[3-2H]nortropine
[3-2H]norpseudotropine
a
Exhibited at c. 100% enrichment.
Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014

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epimerization is seen for tropine but only a very low level for
pseudotropine. When the nor-series is exhibited, similar data were
obtained, although, surprisingly, retention from [3-2H]nortropine
was higher than from [3-2H]tropine. The experiments were also
carried out in the presence of TBON but, as can be seen, this
compound had no influence on the level of incorporation into (4),
endorsing in vivo the lack of impact of this compound as an
inhibitor of the Pseudomonas AT3 TR in vitro.
These findings confirm that, although the metabolism of these
compounds is mainly direct without interconvertion, between 15
and 30% oxidation of the endo-compounds (tropine, nortropine)
takes place, consistent with the quantitative analysis presented in
Section 2.1. However, it cannot be deduced whether the
(nor)tropinone thus formed is then reduced to the same
compound or to a mixture of epimers. Since it has been shown
(see Section 2.2) that nortropinone can be converted to both
forms, either explanation is compatible with the observed loss of
2H.
and 6 h there is a small but significant enrichment of the
norpseudotropine (Fig. 5a). As metabolism selects in favour of
the heavier isotope, this cannot be due to isotopic fractionation,
but rather indicates that this pool is having 15N added to it from
the more enriched nortropine pool.
On the basis of the experiments described above concerning
the retention of 2H from [3-2H]-labelled substrates, between 15
and 20% of the nortropine could be being converted to
norpseudotropine (Table 2). We have therefore constructed a
model with which to calculate the d15N values of the two
substrates at any time of the reaction (see Supplementary
Information). The model requires knowledge of the 15N KIEs for
each existing degradation reaction and these have been obtained
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
´
for tropine N-demethylation (Molinie et al., 2009) and for
´
nortropine ring-scission (Kwiecien et al., 2012) by theoretical
calculation. The reaction flux can be summarized as illustrated in
Fig. 6.
The first hypothesis that can be tested is the situation wherein
the nortropine is the preferred substrate and is consumed first,
with the formation of some norpseudotropine by epimerization
that is added to the initial norpseudotropine pool.
296
297
2.4. Analysis of the interconvertion of nortropine and
norpseudotropine using substrates of differing 15N/14N ratios
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
In order to probe further the implicit double metabolic option
for nortropine degradation (directly and via norpseudotropine)
experiments were conducted in which cells were provided a
mixture of nortropine and norpseudotropine with differing
natural-abundance 15N/14N ratios. By following the evolution of
the isotopic deviation d15N (%) for each of the substrates it was
possible to evaluate how much 15N was metabolized by each route.
The experiments were performed with a mixture of nortropine
In this scenario, any conversion of nortropine to norpseudo-
tropine should both increase the size of the pool and increase the
observed d15N value. However, this hypothesis can quickly be
discarded because it is seen that even in the early hours of the
reaction there must be norpseudotropine consumption, since there
is a small but significant drop in the pseudotropine concentration
(Fig. 5b). Nevertheless, even in conditions in which c. 20% of the
nortropine is taken as being converted to norpseudotropine, it is
still evident that nortropine is preferentially metabolized (see Fig.
SI1a and b).
34345
346
347
348
349
350
351
352
353
354
355
356
(1.73 mM, d15N (%) = 25.6) and norpseudotropine (2.83 mM, d15
(%) = 0.8).
N
The metabolic profile observed is illustrated in Fig. 3 (for
data, see Table SI1): the concentration of nortropine dropped by
90% over the first 6 h, followed by disappearance of the
norpseudotropine. During the first 6 h, the d15N of nortropine
decreases (Fig. 5 a), indicating a strong 15N kinetic isotope effect
In the second scenario, the two substrates are envisaged as
consumed in parallel but at different rates.
(
15N KIE) in favour of the heavier isotope. This is in agreement
To test this hypothesis, the model (see Supplementary
Information) can be used to calculate the value of the isotopic
deviation of norpseudotropine at time t of culture integrating the
transfer rate of nortropine to norpseudotropine:
35789
360
361
with theoretical studies of this metabolism, which indicate there
to be an inverse 15N KIE for ring opening of nor(pseudo)tropine
(Kwiecien et al., 2012). Similarly, between 6 and 9 h the d15N of
´
362
363
norpseudotropine decreases, consistent with the metabolism of
NPSTRI
NTRI
d
ð
15NÞNPSTRI
¼
d
ð
15NÞNPSTRIðtÞ þ
d
ð
15NÞNPSTRIðtÞ
´
this compound by a similar mechanism (Kwiecien et al., 2012).
(It should be noted that after 5 h for nortropine and 9 h for
norpseudotropine insufficient compound remains to obtain a
measurement of the d15N (%).) Critically, however, between 0
For this model, the 15N value of norpseudotropine is thus is the
sum of the contribution of the original isotope 15N value and
36
6
45
6
367
Fig. 6. Metabolic flux for the mixture of co-substrates: nortropine and norpseudotropine. EIC, equilibrium isotope effect; KIE, kinetic isotope effect.
Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014

G Model
PHYTOL 703 1–9
K. Kosieradzka et al. / Phytochemistry Letters xxx (2014) xxx–xxx
7
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383
the15N value of the nortropine-derived norpseudotropine. Apply-
ing this model to the experimental data for the period of co-
metabolism between 0 and 5 h a theoretical curve for the evolution
of the 15N value of norpseudotropine is obtained (Fig. 5c) which
may be compared with the observed 15N values. The closest
approximation of the experimental data is obtained when taking a
conversion rate of 18%, which is within the range of conversions
indicated from the (3-2H data). Furthermore, after the system
switches from co-metabolism to metabolism of norpseudotropine
alone (after 5–7 h of culture), the proposed model fitted with a rate
of nortropine transformation into norpseudotropine of 0% is still
appropriate (Fig. 5c). In addition, it is possible to estimate the
kinetics of the actual consumption of substrates depending on
time. This shows that nortropine is clearly the preferred substrate:
after 5 h of culture, 70% of the nortropine is consumed and only 10%
of the norpseudotropine (Fig. SI1a).
pale yellow crystals from aqueous MeOH (yield 46%). Purity and 431
structural identity were confirmed by 1H NMR. A fresh sample of 432
TBON was prepared each time it was used. 8-Thiabicyclo[3.2.1]oc- 433
tan-1-ol (TBOL) was obtained by the reduction of TBON in MeOH 434
with NaBH4. Following chromatography on a Si gel (40–63
column eluted with diethyl ether/petroleum ether (4:1), -TBOL 436
and -TBOL were obtained as white powders with yields of 9% and 437
30%, respectively. 438
mm) 435
a
b
[3-2H]NortropineꢀHCl and [3-2H]norpseudotropineꢀHCl were 439
prepared by the reduction of nortropinoneꢀHCl with NaB2H4 and 440
separation of the N-carboxylic acid tert-butyl esters. To the crude 441
mixture was added di-tert-butyl dicarbonate in solution in CH2Cl2. 442
Following stirring at room temperature a crude mixture of N- 443
carboxylic acid tert-butyl ester-[3-2H]nortropine and N-carboxylic 444
acid tert-butyl ester-[3-2H]norpseudotropine was obtained. Sepa- 445
ration of the two N-carboxylic acid tert-butyl esters was achieved 446
by column chromatography on silica gel 60 eluted with a gradient 447
of diethyl ether:petroleum ether (50:50–10:90). [3-2H]Nortropi- 448
neꢀHCl and [3-2H]norpseudotropineꢀHCl were recovered by treat- 449
ing the appropriate N-carboxylic acid tert-butyl ester in solution in 450
384
3. Conclusions
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
It is apparent that the interconversions of alkaloid substrates
exploited for growth of Pseudomonas AT3 cultures is complex. In
view of the flexibility of Pseudomonas metabolism this is not
surprising. These results show that there is a metabolic exchange
between nortropine and norpseudotropine under the control of the
TR oxidoreductase activity. Substrate competition experiments
showed that nortropine is the preferred substrate, while labelling
experiments indicated that around 15–20% of nortropine is
converted to norpseudotropine but not significantly vice versa.
This is supported by the very low activity of TR for the formation of
nortropinone from norpseudotropine. It is further supported by the
correlation between the calculated d15N (%) that should be
obtained for an 18% interconvertion and the observed experimen-
CH2Cl2 with HCl gas.
451
To obtain samples of nor(pseudo)tropine slightly impoverished 452
or enriched in 15N, fractionation during column chromatography 453
was exploited as described previously (Kosieradzka et al., 2010). 454
4.2. Bacterial cultures
455
Pseudomonas strains AT3 and MS2 were obtained from Dr P.W. 456
Trudgill (Department of Biochemistry, University of Wales, 457
Aberystwyth, UK). The bacteria were stored at ꢁ80 8C in sterile 458
defined mineral salt medium pH 7.2 containing 15% (v/v) glycerol 459
and cultured in defined mineral medium as described previously 460
(Kosieradzka et al., 2010). For routine culture, 90 mL medium was 461
tal values, using a 15N KIE calculated theoretically (Kwiecien et al.,
´
autoclaved in
Substrate solutions were prepared at c. 70 mM in medium, 463
adjusted to pH 7.2 and sterilized by filtration (0.22 m porosity). 464
a 500 mL Erlenmeyer flask (121 8C; 20 min). 462
2012).
In summary, it can be concluded that:
m
Routinely, 10 mL was added aseptically to 90 mL medium. 465
Bacterial culture was initiated by resuspending frozen culture in 466
sterile defined minimal mineral salt medium pH 7.2. Metabolism 467
was induced by a 10-h pre-culture (30 8C; 200 rpm; obscurity) on 468
tropine (8.0 mM). Once the culture optical dispersion at 550 nm 469
(OD550) was between 0.6 and 0.9, cells were harvested by 470
centrifugation (4500 ꢂ g, 10 min, 4 8C), washed in 100 mL fresh 471
medium without substrate, centrifuged (4500 ꢂ g, 10 min, 4 8C), 472
40
404
40
40
409
41
0
2
3
-
The growth rate of Pseudomonas AT3 is equivalent on both
nortropine and norpseudotropine.
Nortropine is preferred as substrate versus norpseudotropine.
Norpseudotropine is metabolized without significant conversion
to nortropine.
In the presence of both substrates, about 18% of the nortropine is
converted to norpseudotropine, as indicated by both 3-2H-
labelling and changes in the d15N (%) values.
05
07
6
8
-
-
10
1
-
412
413
then resuspended in 15 mL fresh medium.
473
To fresh medium containing substrate (nortropine, norpseudo- 474
tropine, nortropinone, c. 7 mM) as sole carbon and nitrogen source 475
was added an aliquot of bacterial suspension such that the initial 476
OD550 ꢃ 0.2 (c. 1.5 mL). An initial sample (10 mL) was taken at 477
t = 0 h and the cultures were incubated in standard conditions 478
(30 8C; 200 rpm; obscurity). At time points between 0 and 25 h, 479
further samples (5 mL) were taken, the OD550 measured, bacterial 480
cells removed by centrifugation (4500 ꢂ g, 10 min, 4 8C), and the 481
supernatant frozen at ꢁ20 8C until required. Culture growth was 482
followed by OD550, alkaloid consumption by GC (see Section 4.6), 483
414
4. Experimental
415
416
For details of the procedures described concisely in this section,
see Kosieradzka (2010) and Kosieradzka et al. (2010).
417
4.1. Chemicals
418
419
420
421
422
423
424
425
426
427
428
429
430
Nortropine and nortropinoneꢀHCl were obtained from Boeh-
ringer Ingelheim (www.boehringer-ingelheim.com), tropine and
4-hydroxy-N-methylpiperidine from Sigma–Aldrich (www.sig-
maldrich.com), ethyl chloroformate (purity >99%) from Janssen
Chimica (www.acros.com). All other chemicals were of analytical
grade. Pseudotropine and norpseudotropine were synthesized as
described previously (Kosieradzka et al., 2010).
8-Thiabicyclo[3.2.1]octan-1-one (TBON) was synthesized as
described previously (Parr et al., 1991). Briefly, tropinone was
reacted with iodomethane in dry EtOH to give tropinone
methiodide, which was recovered by precipitation in cold diethyl
ether (yield 96%). Tropinone methiodide was reacted with Na2SO4
under an N2 atmosphere to yield TBON, which was crystallized as
and d15N (%) by EA-irm-MS (see Section 4.7).
484
4.3. Alkaloid extraction
485
Culture stored at ꢁ20 8C was rapidly thawed and vacuum 486
filtered through a nitrocellulose filter (pore size, 0.22 m) to 487
m
remove any residual cellular debris. The filtrate was acidified (pH c. 488
2) and applied to a preconditioned (washed with 5 mL of MeOH 489
and 5 mL of neutral water) solid phase ion-exchange extraction 490
cartridge (500 mg Discovery SCX, Supelco, www.sigmaaldrich.- 491
com) fitted to a Visiprep1 vacuum elution manifold (Supelco). 492
Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014

G Model
PHYTOL 703 1–9
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K. Kosieradzka et al. / Phytochemistry Letters xxx (2014) xxx–xxx
493
494
495
496
497
Sample was introduced by low suction, the cartridge rinsed with
neutral water (2 mL) and dried with a flow of N2 gas for 60 min
using the Visidry1 system. Compounds were recovered in MeOH/
NH3 soln. (94:6, v/v, 6 mL). Samples were taken to dryness at 40 8C
with a stream of N2 gas.
4.7. Determination of isotopic enrichment in [2H]-6-
hydroxycyclohepta-1,4-dione
553
554
Pseudomonas MS2 was cultured as described in Section 4.2 for
strain AT3 except that a mixture of tropic acid (3.0 mM) and
tropine (4 mM) was used as substrate for pre-culture. For the
analysis of 2H incorporation into [2H]-6-hydroxycyclohepta-1,4-
dione, cultures (45 mL) were exhibited a mixture of tropic acid
(3.0 mM) and either [3-2H]nortropine (4 mM) or [3-2H]norpseu-
dotropine (4 mM). Once growth attained OD550 ꢃ 0.7, supernatant
was recovered by centrifugation (4500 ꢂ g, 10 min, 5 8C), acidified
(pH = 5.5) with 2 M HCl and extracted with ethyl acetate (5ꢂ
50 mL). The combined organic phases were dried (MgSO4), filtered
and taken to dryness.
The residue was dissolved in ethyl acetate and isotopic
enrichment in [2H]-6-hydroxycyclohepta-1,4-dione was deter-
mined by GC–MS on an Thermo Scientific DSQII single quadrupole
MS system interfaced to a Thermo Scientific Trace GC Ultra fitted
with an HP-5 chromatography column. Standard conditions were:
carrier gas, helium at 1.2 mL minꢁ1 (constant flow, 24 cm/s); split
ratio, 1:40; column HP-5 (30 m, i.d. 0.32 mm, film thickness,
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
498
4.4. Measurement of oxidoreductase activity
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
Pseudomonas AT3 was grown with tropine as a substrate to
OD550 ꢃ 1. Cells were harvested by centrifugation (4500 ꢂ g,
10 min, 5 8C), the pellet was washed twice with sodium phosphate
buffer (100 mM, pH = 7.0, 10 mL) and the cells suspended in the
same buffer containing dithiothreitol (1 mg mLꢁ1). Cell disruption
was carried out by ultrasonication (3ꢂ 15 W, 3 min; pulse 5 s,
4 8C). Debris was removed by centrifugation (4500 ꢂ g, 10 min,
5 8C) followed by filtration (cellulose acetate, 0.2
mm). A fresh
extract was prepared daily.
Enzyme activity was determined by following the change in
OD343 due to the NADP+/NADPH oxidoreduction. Sample was
prepared in sodium phosphate buffer (250 mM pH 6.4, 0.68 mL), to
which was added sequentially cell extract (0.1 mL), water (0.1 mL)
(or inhibitor TBON or
b-TBOL if appropriate, 0.1 mL, 70 mM),
0.25
m
m), injector temperature, 240 8C, detection by FID at 260 8C;
L. The chromatogram was devel-
substrate solution (c. 80 mM, 0.1 mL), cofactor solution (1.3 mM,
0.02 mL). Initial rate of activity was measured over 6–8 min at
30 8C.
Protein concentration was determined from the measured
OD260 and OD280 values (Warburg and Christian, 1942).
injected volume (manual), 1
m
oped using a thermal gradient: 80 8C for 1 min; 10 8C minꢁ1 to
150 8C, 15 8C minꢁ1 to 260 8C, 260 8C for 4 min. Under these
conditions, the retention time was 9.62 min.
4.8. Determination of d15N (%) by irm-MS
578
518
4.5. Measurement of products of oxidoreductase activity
For pure compounds and standards, the d15N (%) value was
obtained from the 15N/14N ratios determined by EA-irm-MS and for
mixtures the d15N (%) values were obtained by GC-irm-MS, both
as described previously (Kosieradzka et al., 2010).
579
580
581
582
519
520
521
522
523
524
525
526
In a tube (15 mL) was mixed sodium phosphate buffer
(250 mM, pH = 6.4, 0.8 mL), glucose dehydrogenase (3.8 U/mg,
0.1 mL), NADP+ (1.3 mM, 0.1 mL), NADPH (1.3 mM, 0.1 mL) and
glucose (0.5 M, 0.1 mL) After 5 min equilibration at 30 8C, cell
extract (0.2 mL) was added followed by substrate (nortropinone or
tropinone) (c. 80 mM, 0.2 mL). The mixture was incubated for 6 h
at 30 8C with gentle stirring. Alkaloids were recovered as described
in Section 4.3 and analyzed by GC as described in Section 4.6.
Acknowledgements
583
We thank Dr P.W. Trudgill (Department of Biochemistry,
University of Wales, Aberystwyth, UK) for supplying us with the
Pseudomonas strains. We thank Ana¨ıs Fournel for the preparation
of the [3-2H]nor(pseudo)tropine and the project ANR-08-PCVI-
584
585
586
587
527
4.6. Quantification of alkaloids by GC
0017 (ISOMODTS) for financing her work, Claire Mayant, (MSc, Q3 588
528
529
530
531
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535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
Quantification was carried out following reaction with ethyl-
chloroformate to form the ethylcarbamate esters using a gas
chromatograph fitted with a ZB WAX column (Phenomenex) and
calibrated with pure standards, as described previously (Kosier-
adzka et al., 2010). Briefly, growth medium (10 mL) was made
alkaline (solid Na2CO3 to 10%, w/v) and ethyl chloroformate (ECF)
in CH2Cl2 (20%, v/v) added (2.5 mL). Following vigorous shaking
(10 min), solid Na2CO3 was added to saturation and extraction
carried out with EtAc (4ꢂ 2 mL). The collected organic phases were
dried and evaporated. For GC, samples were dissolved in MeOH
containing 4-hydroxy-N-methylpiperidine (4NMP) at 4 mg mLꢁ1
as internal reference. Quantification was carried out using the
following standard conditions: chromatograph, Agilent 6890
(www.agilent.com); carrier gas, helium at 1.0 mL minꢁ1; injection
mode, split (1:40); column, cross-linked polyethylene glycol (ZB
EBSI), Begon˜a Rodrigo-Llodio (ERASMUS student, University of
Castilla-La Mancha, Spain) and Carmen Urbina (ERASMUS student,
University of Rioja, Spain) for their contributions to this study.
589
590
591
Appendix A. Supplementary data
592
Supplementarymaterialrelatedtothisarticlecanbefound, inthe
online version, at http://dx.doi.org/10.1016/j.phytol.2014.04.014.
593
594
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Please cite this article in press as: Kosieradzka, K., et al., Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange
between the nortropine and norpseudotropine catabolic pathways. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phy-
tol.2014.04.014