fems microbiology letters volume 145 issue 2 1996 [doi 10.1016%2fs0378-1097%2896%2900420-x] dale r....

8/10/2019 FEMS Microbiology Letters Volume 145 Issue 2 1996 [Doi 10.1016%2Fs0378-1097%2896%2900420-x] Dale R. Walters; Tracy Cowley -- Formation of Cada

1/5

ELSEVIER

FEMS Microbiology Letters 145 (1996) 255-259

Formation of cadaverine derivatives in Saccharomyces cerevisiae

Dale R. Walters *, Tracy Cowley

Department f Plant Science, The Scottish Agricul tural College Auchincruive Nr Ayr KA6 SHW lJK

Received 9 September 1996; accepted 4 October 1996

bstract

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

formed by a wild-type strain of

Saccharomyces cerevi siae,

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 a-

difluoromethylomithine (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;

Saccharomyces cerevisiae

1

IntrodRctlon

The diamine putrescine, the triamine spermidine

and the tetraamine spermine, collectively known as

polyamines, are important for the growth and devel-

opment 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 amino-

propyl groups (NH(CHz)s) from decarboxylated S-

adenosylmethionine (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: [email protected]

aminopropyl additions to putrescine catalysed suc-

cessively by the aminopropyltransferase enzymes

spermidine synthase and spermine synthase [2].

ODC can be inhibited by the suicide inhibitor a-di-

fluoromethylornithine (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 cat-

alysed by lysine decarboxylase or by ODC [3]. Ami-

nopropylation of cadaverine leads to the formation

of the higher homologues aminopropylcadaverine

(APC) and NJ-bis(3-aminopropyl)cadaverine

(3APC). These compounds have been reported pre-

viously in bacteria [4], human tumour cells [5], Nar-

rosparu crussu

[6] and in a number of mycorrhizal

and plant pathogenic fungi [7]. Previous work has

0378-1097/96/. 12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

PZISO378-1097(96)00420-X


2/5

256

D. R. Walters. T. Cowley FEMS Microbiology Letters 145 1996 255-259

Table I

In vitro incorporation of [U-Clornithine into putrescine, sper-

midine and spermine in wild-type S.

cerevisiae

and the mutants

spe 3-l and spe 4-1

Strain

Radioactivity in polyamine

(dpm [mg protein]-)

Putrescine Spermidine Spermine

Wild-type

25.1 48.3 169.2

qx? 3-l

65.3 9.4 185.9

SPt? 4-1

13.0 31.7 9.1

LSD (I= 0.05)

8.7 5.2 12.8

suggested that most fungi synthesise the higher

homologues of cadaverine using AdoMetDC and

the aminopropyltransferases, although some evi-

dence for the operation of a route from L-aspartic-

P-semialdehyde was presented [7]. Here we report on

the formation of cadaverine derivatives in the yeast

Saccharomyces cerevi siae, using mutants with point

mutations for spermidine synthase and spermine

synthase to aid elucidation of the biosynthetic path-

ways involved.

2. aterials and methods

2.1. Growt h condit ions

The S.

cerevisiue

strains Y235 and Y390 were ob-

tained 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 sper-

mine synthase gene (spe 4-l). The yeast strains used

in this work were grown in YPAD medium (1%

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

and 0.04% adenine sulfate). All cultures were incu-

bated 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 spec-

trophotometer.

2.2. In vi tro incorporation of /UL 4CJly sine int o

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 contain-

ing 50 mM Tris HCl (pH S.O), 0.5 mM EDTA and

5 mM DTT and sonicated. The extract was centri-

fuged 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 de-

scribed 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 de-

scribed previously [7]. The in vitro incorporation of

L [l

14C]ornithine into putrescine, spermidine and

spermine, and the in vitro incorporation of

L [U

i4C]aspartic acid plus lysine into cadaverine, APC

Table 2

In vitro incorporation of [U-r4C]lysine into cadaverine, APC and 3APC in wild-type S.

cerevisiae

and in the mutants spe 3-l and ape 4-1,

i: 1 mM MGBG+3 mM CHA

Strain

Wild-type 41.7

61.6

419.2

Wild-type+MGBG and CHA

22.1 180.2

14.2

Spe 3-l 38.9 65.8

521.8

spe 3-l+MGBG and CHA

11.7 128.1

98.3

spe 4-l 43.4 62.1

581.1

ape 4-l+MGBG and CHA

12.5 40.7

278.3

LSD (P = 0.05) 5.3 6.2

38.2

Radioactivity in polyamine (dpm [mg protein]-)

Cadaverine APC 3APC


3/5

Table 3

D.R Wait ers, T. Cow leyl FEM S M icrobi olo gy Lett ers 145 (19 ) 255-259

251

In vitro incorporation of [U-14C]lysine into cadaverine, APC and 3APC in wild-type S. cerevisiae and in the mutants spe 3-1 and spe 4-1,

+ 5 mM DFMO

Strain

Radioactivity in polyamine (dpm [mg protein]-)

Cadaverine

APC 3APc

Wild-type 37.0

63.7

318.1

Wild-type+5 mM DFMO

5.5

29.4 136.0

spe 3-1

40.8

72.1 548.9

spe 3-I+5 mM DFMO 4.1 14.6 192.4

spe

-1 38.7

67.8

518.2

spe

4-1+5 mM DFMO

4.5 49.5 160.5

LSD (P = 0.05)

1.5 8.1

25.6

and 3APC were performed as described previously

[7,9]. Results are the means of five replicates and

all experiments were repeated twice. Statistical sig-

nificance was assessed using least significant differ-

ence.

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).

3.

Results and discussion

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.

cerevi siae.

There was an 81% reduction

in the flux of label from ornithine through to sper-

midine in Y235 compared to the wild-type, suggest-

ing that this qe 3-1 mutant possesses some spermi-

dine 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 of

[U-14C]lysine into cadaverine, APC and 3APC in wild-type S.

cerevistie and in the yeast mutants spe 3-1 and spe 4-l

Strain

Radioactivity in polyamine

(dpm [mg protein]-)

Cadaverine APC 3APC

Wild-type 28.9 59.8 411.0

Wild-type, 37C

3.1

48.2

1053.2

spe 3-l 46.9 69.1 526.1

spe 3-1, 37C

30.2 216.8 1494.9

spe 4-1

35.2 65.1 544.0

spe 4-1, 37oc 21.0

123.3

1394.0

LSD (P = 0.05)

8.1 7.2

29.8

There was considerable flux of label from [U-

14C]lysine through to cadaverine, APC and 3APC

in the three strains of S.

cerevi sia e,

with most of

the label appearing in 3APC (Table 2). The biosyn-

thesis of the cadaverine derivatives is thought to oc-

cur predominantly in the same way as that of sper-

midine and spermine, i.e. by the addition of an

aminopropyl group to cadaverine and APC, to

form APC and 3 APC respectively [7]. These reac-

tions 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 in-

hibit 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


4/5

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 ori-

ginally described as a powerful inhibitor of putres-

tine sensitive mammalian and yeast AdoMetDC [lo].

Nevertheless, the data suggest that either the inhibi-

tion 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-p-

semialdehyde is the aminopropyl donor to putrescine

for spermidine biosynthesis. In this scheme, L-aspar-

tic-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 aminopropyl-

transferases. 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 de-

rivatives (data not shown).

Incorporation of label from [14C]lysine into cada-

verine and its higher homologues was greatly re-

duced 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 37 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 Ado-

MetDC and spermidine synthase. Further, in S. cer-

evisiae exposed to 37C for 2 h, formation of the

cadaverine derivatives from [U-14C]aspartate plus ly-

sine, 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 follow-

ing exposure

to elevated temperatures requires

further investigation. This appears to be the first re-

port of increased formation of the higher homolo-

gues of cadaverine following exposure to elevated

temperature, although it is well known that thermo-

philic 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. cere-

visiae 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 opera-

tion 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.

cknowledgments

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, En-

vironment and Fisheries Department.

References

[I]

Tabor, C.W. and Tabor, H. (1985) Polyamines in microorgan-

isms. Microbial. Rev. 49, 81-99.

[2] Walters, D.R. (1995) Inhibition of polyamine biosynthesis in

fungi. Mycol. Res. 99, 129-139.

[3] Igarashi, K., Kashiwagi, K., Hamasaki, H., Miura, A., Kake-

gawa, T.. Hirose, S. and Matsuzaki, S. (1986) Formation of a

compensatory polyamine by Escherichia cd polyamine-re-

quiring mutants during growth in the absence of polyamines.

J. Bacterial. 166, 128-134.


5/5

D. R. Wal ters, T Cow leyl FEMS M icrobiol ogy Lett ers 145 (1996) 255-259

259

[4] Hamana, K., Niitsu, M., Samejima, K. and Matsuzaki, S.

(1988) Occurrence of aminopropylcadaverine and its amino-

propylderivatives aminopentylnorspermidine and N,N-bis(3-

aminopropyl)cadaverine in Halococcus

acetoinfasciens.

FEMS Microbial. Lett. 50, 79-83.

[5] Alhonen-Hongisto, L. and Janne, J. (1980) Polyamine deple-

tion induces enhanced synthesis and accumulation of cadaver-

ine in cultures of Ehrlich ascites carcinoma cells. Biochem.

Biophys. Res. Commun. 93, 1005-1013.

[6] Paulus, T.J., Kiyonu, P. and Davis, R.H. (1982) Polyamine

deficient Neurospora crassa mutants and synthesis of cadaver-

ine. J. Bacterial. 152, 291-297.

and Physiology of Polyamines in Plants (Slocum, R.D. and

Flores, H.E., Eds.) , pp. 230-234, CRC Press, Boca Raton, FL.

[9] Walters, D.R., Keenan, J.P., Cowley, T., McPherson, A. and

Havis, N.D. (1995) Inhibition of polyamine biosynthesis in

Phytophthora infestans

and

Pyth ium t& mum.

Plant Pathol.

44, 80- 85.

[7] Zarb, J. and Walters, D.R. (1994) The formation of cadaver-

ine, aminopropylcadaverine and N, N-bis(3-aminopropyl)cada-

verine in mycorrhizal and phytopathogenic fungi. Lett. Appl.

Microbial. 19, 277-280.

[lo] Williams-Ashman, H.G. and Schenone, A. (1972) Methyl-

glyoxal bis(guanylhydrazone) as a potent inhibitor of mam-

malian and yeast S-adenosylmethionine decarboxylases. Bio-

them. Biophys. Res. Commun. 46, 2888295.

[II] Tait, G.H. (1976) A new pathway for the biosynthesis of

spermidine. Biochem. Sot. Trans. 4, 610-612.

[12] Holtta, E. and Pohjanpelto, P. (1983) Polyamine starvation

causes accumulation of cadaverine and its derivatives in a

polyamine-dependent strain of Chinese-hamster ovary cells.

Biochem. J. 210, 9455948.

[8] Smith, M.A. (1991) Chromatographic methods for the identi-

[13] Oshima, T. (1983) Unusual polyamines in an extreme thermo-

fication and quantification of polyamines. In: Biochemistry

phile,

Thermus thermophilus.

Adv. Polyamine Res. 4, 4799484.

fems microbiology letters volume 145 issue 2 1996 [doi 10.1016%2fs0378-1097%2896%2900420-x] dale r....

Documents