FEMS Microbiology Letters 158 (1998) 51^55

Distribution of cyclic imide-transforming activity

in microorganisms
Chee-Leong Soong a , Jun Ogawa a , Harmastini Sukiman b , Titik Prana b ,
Made Sri Prana b , Sakayu Shimizu a; *
a
Department of Agricultural Chemistry, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-01, Japan
b
R and D Center for Biotechnology, Indonesian Institute of Sciences, Cibinong 422 Bogor, Indonesia

Received 17 September 1997; revised 17 October 1997; accepted 28 October 1997

Abstract

Cyclic imide-transforming activity was found to be widely distributed in bacteria, yeast and molds. This activity was not
correlated with cyclic ureide-transforming activity in bacteria, but there was some correlation in yeast and molds. These two
activities are probably catalyzed by different enzymes in bacteria. Besides the well-known cyclic ureide transformation, cyclic
imide transformation by microorganisms was common.
ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

Keywords : Cyclic imide ; Cyclic ureide; Imidase ; Dihydropyrimidinase ; Hydantoinase

1. Introduction volve dihydropyrimidine [3] and hydantoin [4] deg-

radation pathways. The ring-opening of the ¢rst
The cyclic imide transformation (Fig. 1A) pathway compound is catalyzed by dihydropyrimidinase and
was ¢rst reported in a bacterium Blastobacter sp. the latter by hydantoinase. Some hydantoinases have
A17p-4 [1]. This transformation involves ring-open- been reported to be identical with dihydropyrimidi-
ing hydrolysis of cyclic imides catalyzed by a novel nase [5,6], and play important roles in pyrimidine-
enzyme, imidase [2], successive amidohydrolysis of base metabolism.
half-amide, and further transformation through Yamada et al. [7] reported that the hydantoin hy-
TCA cycle-like reactions. The existence of this kind drolyzing activity is mainly distributed in bacteria
of transformation pathway in other microorganisms and is related to dihydropyrimidine hydrolyzing ac-
has not yet been examined. However, the transfor- tivity. However, to our knowledge, neither the dis-
mation of cyclic ureides (Fig. 1B and C), which have tribution of cyclic imide transformation in microor-
a structure similar to cyclic imides, has been reported ganisms nor this transformation in comparison with
in some microorganisms. These transformations in- those of cyclic ureides has been reported. We thus
studied the distribution of cyclic imide transforma-
* Corresponding author. Tel.: +81 (75) 753-6115; tion in microorganisms, and compared it with cyclic
Fax: +81 (75) 753-6128. ureide transformation.

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 9 9 - 0

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ugation at 14 000Ug for 10 min, and washed twice

with physiological saline and then centrifuged. These
cells were then suspended in 20 mM Tris/HCl,
pH 7.5 and used as washed cells. Mold cells were
harvested by ¢ltration, and the cell-free extracts
were prepared by grinding the cells in a mor-
tar with a pestle in the presence of 1% (w/v) sea
sand B and a small volume of 20 mM Tris/HCl,
pH 7.5. The disrupted mold cells were then cen-
trifuged at 14 000Ug for 120 min and the superna-
tant was used as the cell-free extract. The protein
Fig. 1. The transformation pathways for cyclic imides (A), dihy- concentration was measured by the Bradford meth-
dropyrimidines (B), and hydantoins (C). (1) imidase, (2) half-ami-
dase, (3) dihydropyrimidinase, (4) L-ureidopropionase, (5) hydan-
od.
toinase, (6) N-carbamoyl amino acid amidohydrolase.
2.4. Reaction with intact cells and cell-free extracts

2. Materials and methods Succinimide, dihydrouracil and DL-5-methylhydan-

toin were used as substrates to determine imidase,
2.1. Chemicals and microorganisms dihydropyrimidinase and hydantoinase activities, re-
spectively, in bacteria, yeasts and molds. Each reac-
DL-5-methylhydantoin was a kind gift from Kane- tion mixture consisted of 1% (wt/vol) washed cells or
gafuchi Chemical Co. (Takasago, Japan). All other 50 Wl of cell-free extract, 20 Wmol of Tris/HCl (pH
chemicals were of analytical grade and available 7.5), and 4 Wmol of substrate, in a total volume of
commercially. Microorganisms were obtained from 100 Wl. The reaction was allowed to proceed at 30³C
the culture collection of the Faculty of Agriculture, with shaking for 2^6 h for washed cells of bacteria
Kyoto University (AKU). and yeasts, or 7^15 h without shaking for cell-free
extracts of molds. The reaction was stopped with 10
2.2. Medium and culture conditions Wl of 15% (v/v) perchloric acid, followed by neutral-
ization with 90 Wl 500 mM potassium phosphate (pH
The medium for culture of bacteria was composed 7.0), and centrifugation at 10 000Ug for 10 min. The
of 1 g KH2 PO4 , 1 g K2 HPO4 , 0.3 g MgSO4 W7H2 O, supernatant was analyzed for utilization of sub-
3 g yeast extract, 3 g meat extract, 10 g glycerol and strates on a Shimadzu LC-6A HPLC at 210 nm,
2 g peptone in 1 l of tap water, pH 7.0. Medium for ¢tted with a Cosmosil 5C18 -packed column
culture of yeasts contained 50 g glucose, 5 g peptone, (4.6U250 mm; Nacalai Tesque, Kyoto, Japan) run
4 g KH2 PO4 , 2 g K2 HPO4 , 0.2 g MgSO4 W7H2 O and at a £ow rate of 1.0 ml/min, with 250 mM KH2 PO4
2 g yeast extract in 1 l of tap water, pH 6.0. Medium (pH 4.4) as an eluent.
for culture of molds contained 50 g malt extract and
3 g yeast extract in 1 l of tap water, pH 5.6. 2.5. Assimilation of succinimide
Each microorganism was inoculated into a test
tube containing 5 ml of medium and then incubated The assimilation of succinimide as a sole car-
for 2^4 days at 28³C with shaking. The culture was bon source was investigated in microorganisms list-
then transferred to a 2 l £ask containing 500 ml of ed in Fig. 2. Microorganisms were cultivated in
medium and incubated for 2^4 days for bacteria and minimum liquid medium as described previously [1]
yeasts, or 4^7 days for molds, at 28³C with shaking. containing 20 mM succinimide, at pH 7.0 for
bacteria and at pH 6.0 for yeasts and molds. Culti-
2.3. Preparation of intact cells and cell-free extracts vation was carried out at 28³C with shaking for 2^
4 days for bacteria and yeasts, and 4^7 days for
Bacterial and yeast cells were harvested by centrif- molds.

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Fig. 2. Cyclic imide- and cyclic ureide-transforming activities in bacteria, yeasts and molds.

2.6. Investigation of cyclic imide-transforming cinimide, succinamic acid and succinate were used as
activities the substrates.

The successive cyclic imide-transforming activities,

i.e. succinimide-, succinamic acid-, and succinate- 3. Results
transforming activities [1] were investigated in micro-
organisms growing vigorously in minimum liquid 3.1. Cyclic imide- and cyclic ureide-transforming
medium with succinimide as a sole carbon source. activities in bacteria, yeasts and molds
The reaction conditions and analytical methods
were the same as described above, except that suc- Cyclic imide- and cyclic ureide-transforming activ-

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Table 1
Cyclic imide-transforming activities in microorganisms
Microorganism AKU no. Growtha Speci¢c transforming activity towardb
(mg ml31 ) (Wmol (min g of wet cells)31 )
Succinimide Succinamic acid Succinate
Bacteria
Blastobacter sp. A17p-4 990 0.74 264 10.7 2.88
Bacillus subtilis 210 1.4 46.3 12.9 5.32
Arthrobacter sp. J11 634 0.79 3.77 116 23.0
Cellulomonas sp. 672 0.99 12.8 0.510 6.74
Chromobacterium iodinum 814 0.27 11.7 45.9 13.4
Pseudomonas chlororaphis 873 1.7 192 53.6 38.9
Yeasts
Saccharomyces cerevisiae 4136 3.0 3.20 1.16 0.550
Pichia polymorpha 4250 1.9 3.62 1.10 0.750
Candida succiphila 4642 1.7 1.70 2.69 tr
Molds
Penicillium lilacinum 3415 1.4 0.950 0.960 0.240
Fusarium sorani 3710 2.5 0.930 2.10 1.44
Trichoderma hamatum 3563 2.5 0.650 2.06 tr
a
Wet cell weight of bacteria and yeast was calculated from the calibrated OD 610 nm standard curve with three separate determinations that
were reproducible within þ 10%. Wet cell weight of molds was determined directly after harvest by ¢ltration with three separate determi-
nations that were reproducible within þ 10%.
b
Speci¢c activities of cells are averages of three separate determinations that were reproducible within þ 10%.
tr, trace activity.

ities were investigated in 232 bacteria (30 genera), most yeast strains especially in Saccharomyces (Fig.
250 yeasts (23 genera) and 118 molds (29 genera). 2), and also in molds especially in Penicillium (Fig.
Fig. 2 shows the activities of transformation of cyclic 2). These ¢ndings suggest that cyclic imide and cyclic
imide (succinimide), and cyclic ureide (dihydrouracil ureide transformation are correlated in fungi. In oth-
and DL-5-methylhydantoin) transformation in se- er yeast strains such as Saccharomyces marxianus
lected bacteria, yeasts and molds. AKU 4105, Saccharomyces rosei AKU 4107, Schizo-
Among bacteria (Fig. 2), Pseudomonas strains saccharomyces liquefaciens AKU 4222, Pichia treha-
showed high activities for both cyclic imide and cy- lophilia AKU 4260 and Lipomyces lipofer AKU
clic ureide transformation, while Arthrobacter, Agro- 4420; and mold strains such as Aspergillus terreus
bacterium and Brevibacterium strains showed high AKU 3348, Penicillum rubrum AKU 3409, Penicil-
cyclic imide-transforming activity with relatively lum janthinellum AKU 3418, Neurospora crassa
low cyclic ureide-transforming activity. In general, AKU 3555, Syncepalis sphaerica AKU 3690, Fusa-
cyclic imide- and cyclic ureide-transforming activities rium oxysporum AKU 3702, Trichophyton mentagro-
did not correlate in bacteria. Other bacterial strains phytes AKU 3872, Beauveria bassiana AKU 3876,
such as Citrobacter freundii AKU 38, Erwinia caro- Mortierella isabellina AKU 3999-2, and Mortierella
tovora AKU 40, Agrobacterium tumefaciens AKU ramanniana AKU 3999-7, the cyclic imide- and cyclic
305, Micrococcus luteus AKU 543, Arthrobacter ureide-transforming activity was clearly evident.
atrocyaneus AKU 637, Brevibacterium divaricatum
AKU 640, Pseudomonas maltophilia AKU 848 and 3.2. Assimilation of cyclic imides in microorganisms
Pseudomonas putida 77 AKU 875 also showed con-
siderable cyclic imide-transforming activity. Since Blastobacter sp. A17p-4 utilizes succinimide
Yeasts and molds showed lower cyclic imide-trans- as a sole carbon source [1], we examined whether this
forming activity than bacteria. Both cyclic imide- activity also exists in other microorganisms. The
and cyclic ureide-transforming activities coexist in strains listed in Fig. 2 which showed active cyclic

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imide transformation were cultured in the minimum and that cyclic imides can be utilized as an energy
liquid medium with succinimide as a sole carbon source for growth. Since a few natural cyclic imide
source. Several strains listed in Table 1 (bacteria: compounds are known, the physiological role of cy-
Bacillus, Arthrobacter, Pseudomonas; yeasts: Saccha- clic imide-transforming activity may be in degrada-
romyces; molds: Penicillium, Fusarium) showed sig- tion of xenobiotics [9]. The present ¢ndings clearly
ni¢cant growth. This suggested that cyclic imide-as- show that microorganisms can transform cyclic
similating activity exists in various microorganisms. imides.

3.3. Successive cyclic imide-transforming enzyme

activities in microorganisms References

The successive enzyme activities involved in cyclic [1] Ogawa, J., Soong, C.-L., Honda, M. and Shimizu, S. (1996)
Novel metabolic transformation pathway for cyclic imides in
imide transformation were investigated in strains
Blastobacter sp. A17p-4. Appl. Environ. Microbiol. 62, 3814^
which showed signi¢cant growth in minimum liquid 3817.
medium (Table 1). Succinimide-, succinamic acid- [2] Ogawa, J., Soong, C.-L., Honda, M. and Shimizu, S. (1997)
and succinate-transforming activities were generally Imidase, a dihydropyrimidinase-like enzyme involved in the
higher in bacteria than in yeasts and molds, and metabolism of cyclic imides. Eur. J. Biochem. 243, 322^327.
[3] Van Der Drift, C. and Vogels, G.D. (1976) Degradation of
these activities were expressed at di¡erent levels. In
purine and pyrimidine by microorganisms. Bacteriol. Rev. 40,
yeasts and molds, however, the successive transform- 403^468.
ing activities were nearly equivalent. [4] Ogawa, J., Shimizu, S. and Yamada, H. (1993) N-Carbamoyl-
D-amino acid amidohydrolase from Comamonas sp. E222c:
puri¢cation and characterization. Eur. J. Biochem. 212, 685^
691.
4. Discussion
[5] Runser, S., Chinski, N. and Ohleyer, E. (1990) D-p-Hydrox-
yphenyl-glycine production from DL-5-p-hydroxyphenylhy-
In bacteria, no correlation was observed among dantoin by Agrobacterium sp. Appl. Microbiol. Biotechnol.
succinimide-, dihydrouracil- and DL-5-methylhydan- 33, 382^388.
toin-hydrolyzing activities, suggesting that cyclic [6] Takahashi, S., Kii, Y., Kumagai, H. and Yamada, H. (1978)
Puri¢cation, crystallization and properties of hydantoinase
imide and cyclic ureide transformation involve di¡er-
from Pseudomonas striata. J. Ferment. Technol. 56, 492^498.
ent enzyme systems as observed in Blastobacter sp. [7] Yamada, H., Takahashi, S., Kii, Y. and Kumagai, H. (1978)
[10]. This may be common in procaryotes. However, Distribution of hydantoin hydrolyzing activity in microorgan-
the relative activity of cyclic imide transformation to isms. J. Ferment. Technol. 56, 484^491.
that of cyclic ureide transformation was similar in all [8] Maguire, J.H. and Dudley, K.H. (1978) Dihydropyrimidinase,
metabolism of some cyclic imides of di¡erent ring size. Drug
fungi examined. Together with the reports that both
Metab. Dispos. 6, 140^145.
cyclic imides and cyclic ureides are hydrolyzed by [9] Yang, Y.-S., Ramaswamy, S. and Jakoby, W.B. (1993) Rat
mammalian dihydropyrimidinases [8,9], the present liver imidase. J. Biol. Chem. 268, 10870^10875.
¢ndings suggest that these two activities are cata- [10] Ogawa, J., Honda, M., Soong, C.-L. and Shimizu, S. (1995)
lyzed by an identical enzyme system in eucaryotes. Diversity of cyclic ureide compounds, dihydropyrimidine-,
and hydantoin-hydrolyzing enzymes in Blastobacter sp.
In summary, we revealed that cyclic imide-trans-
A17p-4. Biosci. Biotech. Biochem. 59, 1960^1962.
forming activity commonly exists in microorganisms,

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