ELSEVIER

FEMS Microbiology Letters 145 (1996) 255-259

Formation

of cadaverine derivatives in Saccharomyces cerevisiae

Dale R. Walters *, Tracy Cowley

Departmentof Plant Science, The Scottish Agricultural College, Auchincruive, Nr Ayr KA6 SHW, lJK
Received 9 September

1996; accepted 4 October

1996

Abstract

The higher homologues of cadaverine, aminopropylcadaverine (APC) and NJ-bis(3-aminopropyl)cadaverine

(3APC) were
formed by a wild-type strain of Saccharomyces cerevisiae, and by two mutant strains, spe 3-1 and spe 4-1, exhibiting point
mutations in the genes for spermidine synthase and spermine sy-nthase, respectively. This, together with the incomplete
inhibition of APC and 3APC formation in the presence of inhibitors of S-adenosylmethionine decarboxylase and spermidine
synthase, suggests that the cadaverine derivatives are formed partly by the operation of a different route. However, the yeast
strains were unable to utilise [14C]aspartate and lysine to form APC and 3APC. Since the ornithine decarboxylase inhibitor adifluoromethylomithine (DFMO) greatly reduced the formation of APC and 3APC, it is suggested that these compounds are
formed preferentially in these yeast strains from cadaverine formed by ODC. APC and 3APC formation in the yeast strains was
increased substantially following exposure to 37C for 2 h.
Keywords:

Cadavetie;

Aminopropylcadaverine;

N,N-Bis(3-aminopropyl)cadaverine;

1. IntrodRctlon

The diamine putrescine, the triamine spermidine

and the tetraamine spermine, collectively known as
polyamines, are important for the growth and development of all cells [l]. In most fungi, putrescine is
formed by decarboxylation of omithine in a reaction
catalysed by the enzyme ornithine decarboxylase
(ODC), while spermidine and spermine are formed
from putrescine by subsequent additions of aminopropyl groups (NH(CHz)s) from decarboxylated Sadenosylmethionine (AdoMet). The formation of
these aminopropyl groups from AdoMet is catalysed
by AdoMet decarboxylase (AdoMetDC), and the
* Corresponding
author. Tel.: +44 (1292) 525307;
Fax: +44 (1292) 525314; E-mail: d.walters@au.sac.ac.uk
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of European

Saccharomyces cerevisiae

aminopropyl additions to putrescine catalysed successively by the aminopropyltransferase enzymes

spermidine synthase and spermine synthase [2].
ODC can be inhibited by the suicide inhibitor a-difluoromethylornithine
(DFMO), AdoMetDC by
methylglyoxal bis(guanylhydrazone) (MGBG), and
spermidine synthase by cyclohexylamine (CHA) [2].
Decarboxylation of lysine leads to the formation
of the diamine cadaverine. This reaction can be catalysed by lysine decarboxylase or by ODC [3]. Aminopropylation of cadaverine leads to the formation
of the higher homologues aminopropylcadaverine
(APC)
and
NJ-bis(3-aminopropyl)cadaverine
(3APC). These compounds have been reported previously in bacteria [4], human tumour cells [5], Narrosparu crussu [6] and in a number of mycorrhizal
and plant pathogenic fungi [7]. Previous work has
Microbiological

Societies. Published

by Elsevier Science B.V.

D. R. Walters. T. Cowley

256
Table I
In vitro incorporation
midine and spermine
spe 3-l and spe 4-1
Strain

Wild-type
qx? 3-l
SPt?4-1
LSD (I= 0.05)

/ FEMS

of [U-Clornithine
into putrescine, sperin wild-type S. cerevisiae and the mutants

Radioactivity in polyamine
(dpm [mg protein]-)
Putrescine

Spermidine

Spermine

25.1
65.3
13.0
8.7

48.3
9.4
31.7
5.2

169.2
185.9
9.1
12.8

suggested that most fungi synthesise the higher

homologues
of cadaverine using AdoMetDC
and
the aminopropyltransferases,
although
some evidence for the operation of a route from L-asparticP-semialdehyde was presented [7]. Here we report on
the formation of cadaverine derivatives in the yeast
Saccharomyces cerevisiae, using mutants with point
mutations
for spermidine
synthase and spermine
synthase to aid elucidation of the biosynthetic pathways involved.

2. Materials and methods

2.1. Growth conditions
The S. cerevisiue strains Y235 and Y390 were obtained from Dr Celia White Tabor of the National
Institutes
of Health, Bethesda, USA. The strain
Y235 has a point mutation
in the spermidine
synthase gene (spe 3-l), while strain Y390 has point
mutations in the ODC gene (spe 10-l) and the spermine synthase gene (spe 4-l). The yeast strains used
in this work were grown in YPAD medium (1%
Table 2
In vitro incorporation
of [U-r4C]lysine into cadaverine,
i: 1 mM MGBG+3 mM CHA
Strain

Wild-type
Wild-type+MGBG
and CHA
Spe 3-l
spe 3-l+MGBG
and CHA
spe 4-l
ape 4-l+MGBG
and CHA
LSD (P = 0.05)

Microbiology

Letters 145

255-259

yeast extract; 2% peptone; 2% dextrose; 2% agar

and 0.04% adenine sulfate). All cultures were incubated at 30C with rotary shaking and growth curves
obtained by measuring the optical density of the
cultures at 600 nm in a Gallenkamp Visi-Spec spectrophotometer.
2.2. In vitro incorporation of /UL4CJlysine

into

cadaverine derivatives

Yeast cells (from 20 ml of YPAD medium) were

harvested by centrifugation
at 8000 X g for 15 min
and suspended in 3 ml of extraction buffer containing 50 mM Tris HCl (pH S.O), 0.5 mM EDTA and
5 mM DTT and sonicated. The extract was centrifuged for 15 min at 5000 x g at 4C and 100 ul of the
supernatant
used in a LDC assay. reaction mixture
contained 10 mM Tris HCl (pH 8.0), 1 mM DTT,
0.1 mM EDTA, 0.1 mM pyridoxal phosphate, 5 mM
lysine, 3.7 kBq [U-14C]lysine (11 GBq mmol-,
Amersham International)
and 100 ml of the yeast
extract in a total volume of 250 ~1. The reaction
was initiated by addition of the substrate and was
carried out in test tubes on a shaking water bath for
1 h at 37C. The reaction was terminated by the
addition of 0.2 ml of 6 N HsS04, incubated for a
further 30 min and 100 ml aliquots of the reaction
mixture used for polyamine analysis by TLC as described by Smith [8]. APC and 3APC markers were
run on TLC plates together with the yeast extracts
and the identity of the cadaverine derivatives in the
extracts confirmed by NMR spectroscopy as described previously [7]. The in vitro incorporation
of
L-[l- 14C]ornithine
into putrescine,
spermidine
and
spermine, and the in vitro incorporation
of L-[Ui4C]aspartic acid plus lysine into cadaverine, APC

APC and 3APC in wild-type

Radioactivity

/ 1996)

in polyamine

S. cerevisiae and in the mutants

spe 3-l and ape 4-1,

(dpm [mg protein]-)

Cadaverine

APC

3APC

41.7
22.1
38.9
11.7
43.4
12.5
5.3

61.6
180.2
65.8
128.1
62.1
40.7
6.2

419.2
14.2
521.8
98.3
581.1
278.3
38.2

D.R
Table 3
In vitro incorporation
+ 5 mM DFMO

Waiters, T. Cowleyl FEMS Microbiology Letters 145 (19%) 255-259

of [U-14C]lysine into cadaverine,

APC and 3APC in wild-type

Radioactivity

Strain

Wild-type
Wild-type+5 mM DFMO
spe 3-1
spe 3-I+5 mM DFMO
spe4-1
spe 4-1+5 mM DFMO
LSD (P = 0.05)

3APc

37.0
5.5
40.8
4.1
38.7
4.5
1.5

63.7
29.4
72.1
14.6
67.8
49.5
8.1

318.1
136.0
548.9
192.4
518.2
160.5
25.6

The S. cerevisiae strains Y235 and Y390, with

point mutations in the genes for spermidine synthase
(spe 3-l), and ODC (spe 10-l) and spermine synthase
(spe 4-l), respectively, were examined for polyamine
biosynthetic activity, in comparison with a wild-type
strain of S. cerevisiae. There was an 81% reduction
in the flux of label from ornithine through to spermidine in Y235 compared to the wild-type, suggesting that this qe 3-1 mutant possesses some spermidine synthase activity (Table 1). Probably because of
the incomplete depletion of spermidine, formation of

Table 4
Effects of exposure to 37C for 2 h on in vitro incorporation
[U-14C]lysine into cadaverine, APC and 3APC in wild-type
cerevistie and in the yeast mutants spe 3-1 and spe 4-l

Wild-type
Wild-type, 37C
spe 3-l
spe 3-1, 37C
spe 4-1
spe 4-1, 37oc
LSD (P = 0.05)

Radioactivity in polyamine
(dpm [mg protein]-)
APC

28.9
3.1
46.9
30.2
35.2
21.0
8.1

59.8
48.2
69.1
216.8
65.1
123.3
7.2

(dpm [mg protein]-)

APC

3. Results and discussion

Cadaverine

spe 3-1 and spe 4-1,

Cadaverine

and 3APC were performed as described previously

[7,9]. Results are the means of five replicates and
all experiments were repeated twice. Statistical significance was assessed using least significant difference.

Strain

in polyamine

S. cerevisiae and in the mutants

251

3APC
411.0
1053.2
526.1
1494.9
544.0
1394.0
29.8

of
S.

spermine in this mutant was similar to that observed

in the wild-type strain (Table 1). Strain Y390 showed
reductions in putrescine, spermidine and spermine
formation of 49, 35 and 95%, respectively, indicating
the presence of substantial ODC activity, but very
low spermine synthase activity (Table 1).
There was considerable flux of label from [U14C]lysine through to cadaverine, APC and 3APC
in the three strains of S. cerevisiae, with most of
the label appearing in 3APC (Table 2). The biosynthesis of the cadaverine derivatives is thought to occur predominantly in the same way as that of spermidine and spermine, i.e. by the addition of an
aminopropyl group to cadaverine and APC, to
form APC and 3 APC respectively [7]. These reactions would be catalysed by AdoMetDC and the
aminopropyltransferases, spermidine synthase and
spermine synthase. Yet, in the mutants spe 3-l and
spe 4-1, which appear to possess low spermidine
synthase activity, formation of APC and 3APC
from labelled lysine was little different from the
wild-type (Table 2). It is possible that both of these
mutants possess sufficient aminopropyltransferase
activities to maintain the biosynthesis of cadaverine
derivatives at wild-type levels. In an attempt to inhibit the activities of AdoMetDC and spermidine
synthase and so block the formation of APC and
3APC, the AdoMetDC inhibitor MGBG and the
spermidine synthase inhibitor CHA, were used.
Treatment with MGBG+CHA resulted in a doubling
of APC formation and curiously, an 82% reduction
in formation of 3APC in spe 3-1, while in spe 4-1,
MGBG+CHA reduced formation of cadaverine,
APC and 3APC by 72, 35 and 53%, respectively
(Table 2). It seems unlikely that AdoMetDC from

258

D.R. Walters. T Cowleyl FEMS Microbiology Let&w 145 (1996) 255.-259

the yeast strains used in this study exhibited reduced

sensitivity to MGBG, since this compound was originally described as a powerful inhibitor of putrestine sensitive mammalian and yeast AdoMetDC [lo].
Nevertheless, the data suggest that either the inhibition of enzyme activity by MGBG and CHA was
incomplete, or in addition to AdoMetDC
and the
aminopropyltransferases,
APC and 3APC can also
be formed by another route in S. cerevisiue. Tait
[l l] has shown that in some bacteria, L-aspartic-psemialdehyde is the aminopropyl donor to putrescine
for spermidine biosynthesis. In this scheme, L-aspartic-B-semialdehyde
condenses
with putrescine
to
form a Schiff base, which is then reduced to form
carboxyspermidine,
followed by decarboxylation
to
yield spermidine. Zarb and Walters [7] found that
in the ectomycorrhizal
fungus Laccaria proxima,
APC and 3APC were formed partly by this route
and partly by AdoMetDC
and the aminopropyltransferases. Interestingly, the Schiff base route could
not be detected in the three yeast strains used in the
present work since they were unable to utilise
[14C]aspartate and lysine to form the cadaverine derivatives (data not shown).
Incorporation
of label from [14C]lysine into cadaverine and its higher homologues was greatly reduced in the presence of 5 mM DFMO (Table 3).
This suggests that in these yeast strains, APC and
3APC were formed from cadaverine synthesised by
ODC. This agrees with other work in Escherichia coli
[3] and Chinese hamster
ovary cells [12] which
showed that the higher derivatives of cadaverine
were formed preferentially
from cadaverine made
by ODC.
Exposure of the wild-type S. cerevisiae to 37C for
2 h resulted in a 250% increase in 3APC formation
(Table 4). In spe 3-1, exposure to this mild heat
shock resulted in increases of 310 and 280% in
APC and 3APC formation, respectively, and similar
results were obtained with spe 4-l (Table 4). These
increases in formation of the cadaverine derivatives
were not affected by exposure to MGBG+CHA
(data not shown), suggesting that their formation
under these conditions was not catalysed by AdoMetDC and spermidine synthase. Further, in S. cerevisiae exposed to 37C for 2 h, formation
of the
cadaverine derivatives from [U-14C]aspartate plus lysine, in the presence of MGBG+CHA,
could not be

detected (data not shown). Thus, the increased

synthesis of APC and 3APC following mild heat
shock did not occur via the Schiff base route.
Clearly, the biosynthesis of these compounds followto elevated temperatures
requires
ing exposure
further investigation. This appears to be the first report of increased formation of the higher homologues of cadaverine following exposure to elevated
temperature, although it is well known that thermophilic bacteria synthesise an array of uncommon
analogues of spermidine and spermine when grown
at elevated temperatures [ 131. These compounds, e.g.
norspermidine
(caldine) and norspermine (thermine)
appear to be essential for continued protein synthesis
at high temperatures, both in vivo and in vitro [13].
Whether APC and 3APC fulfill any role in S. cerevisiae grown at elevated temperatures
is not known.
In summary, these data show that S. cerevisiae
forms the higher homologues of cadaverine, APC
and 3APC, and their formation is greatly increased
following exposure to 37C for 2 h. APC and 3APC
formation appears to occur only partly via the action
of AdoMetDC
and the aminopropyltransferases,
and no evidence could be found for the operation of the Schiff base route proposed by Tait
[l 11. Further work is needed to elucidate fully the
route(s) by which these compounds are synthesised
in yeast.

Acknowledgments
We are most grateful to Dr. C.W. Tabor for the
cultures of Y235 and Y390. S.A.C. receives financial
assistance from the Scottish Office Agriculture, Environment and Fisheries Department.

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D. R. Walters, T Cowleyl FEMS

Microbiology

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