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-

The cyclic imide transformation (Fig. 1A) pathway
was ¢rst reported in a bacterium Blastobacter sp.
A17p-4 [1]. This transformation involves ring-open-
ing hydrolysis of cyclic imides catalyzed by a novel
enzyme, imidase [2], successive amidohydrolysis of
half-amide, and further transformation through
TCA cycle-like reactions. The existence of this kind
of transformation pathway in other microorganisms
has not yet been examined. However, the transfor-
mation of cyclic ureides (Fig. 1B and C), which have
a structure similar to cyclic imides, has been reported
in some microorganisms. These transformations in-
* Corresponding author. Tel.: +81 (75) 753-6115;
Fax: +81 (75) 753-6128.
radation pathways. The ring-opening of the ¢rst
compound is catalyzed by dihydropyrimidinase and
the latter by hydantoinase. Some hydantoinases have
been reported to be identical with dihydropyrimidi-
nase [5,6], and play important roles in pyrimidine-
base metabolism.
Yamada et al. [7] reported that the hydantoin hy-
drolyzing activity is mainly distributed in bacteria
and is related to dihydropyrimidine hydrolyzing ac-
tivity. However, to our knowledge, neither the dis-
tribution of cyclic imide transformation in microor-
ganisms nor this transformation in comparison with
those of cyclic ureides has been reported. We thus
studied the distribution of cyclic imide transforma-
tion in microorganisms, and compared it with cyclic
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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52 C.-L. Soong et al. / FEMS Microbiology Letters 158 (1998) 51^55
Fig. 1. The transformation pathways for cyclic imides (A), dihy-
dropyrimidines (B), and hydantoins (C). (1) imidase, (2) half-ami-
dase, (3) dihydropyrimidinase, (4) L-ureidopropionase, (5) hydan-
toinase, (6) N-carbamoyl amino acid amidohydrolase.
2. Materials and methods
2.1. Chemicals and microorganisms
DL-5-methylhydantoin was a kind gift from Kane-
gafuchi Chemical Co. (Takasago, Japan). All other
chemicals were of analytical grade and available
commercially. Microorganisms were obtained from
the culture collection of the Faculty of Agriculture,
Kyoto University (AKU).
2.2. Medium and culture conditions
The medium for culture of bacteria was composed
of 1 g KH2 PO4 , 1 g K2 HPO4 , 0.3 g MgSO4 W7H2 O,
3 g yeast extract, 3 g meat extract, 10 g glycerol and
2 g peptone in 1 l of tap water, pH 7.0. Medium for
culture of yeasts contained 50 g glucose, 5 g peptone,
4 g KH2 PO4 , 2 g K2 HPO4 , 0.2 g MgSO4 W7H2 O and
2 g yeast extract in 1 l of tap water, pH 6.0. Medium
for culture of molds contained 50 g malt extract and
3 g yeast extract in 1 l of tap water, pH 5.6.
Each microorganism was inoculated into a test
tube containing 5 ml of medium and then incubated
for 2^4 days at 28³C with shaking. The culture was
then transferred to a 2 l £ask containing 500 ml of
medium and incubated for 2^4 days for bacteria and
yeasts, or 4^7 days for molds, at 28³C with shaking.
2.3. Preparation of intact cells and cell-free extracts
Bacterial and yeast cells were harvested by centrif-
FEMSLE 7927 5-1-98
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
concentration was measured by the Bradford meth-
od.
2.4. Reaction with intact cells and cell-free extracts
Succinimide, dihydrouracil and DL-5-methylhydan-
toin were used as substrates to determine imidase,
dihydropyrimidinase and hydantoinase activities, re-
spectively, in bacteria, yeasts and molds. Each reac-
tion mixture consisted of 1% (wt/vol) washed cells or
50 Wl of cell-free extract, 20 Wmol of Tris/HCl (pH
7.5), and 4 Wmol of substrate, in a total volume of
100 Wl. The reaction was allowed to proceed at 30³C
with shaking for 2^6 h for washed cells of bacteria
and yeasts, or 7^15 h without shaking for cell-free
extracts of molds. The reaction was stopped with 10
Wl of 15% (v/v) perchloric acid, followed by neutral-
ization with 90 Wl 500 mM potassium phosphate (pH
7.0), and centrifugation at 10 000Ug for 10 min. The
supernatant was analyzed for utilization of sub-
strates on a Shimadzu LC-6A HPLC at 210 nm,
¢tted with a Cosmosil 5C18 -packed column
(4.6U250 mm; Nacalai Tesque, Kyoto, Japan) run
at a £ow rate of 1.0 ml/min, with 250 mM KH2 PO4
(pH 4.4) as an eluent.
2.5. Assimilation of succinimide
The assimilation of succinimide as a sole car-
bon source was investigated in microorganisms list-
ed in Fig. 2. Microorganisms were cultivated in
minimum liquid medium as described previously [1]
containing 20 mM succinimide, at pH 7.0 for
bacteria and at pH 6.0 for yeasts and molds. Culti-
vation was carried out at 28³C with shaking for 2^
4 days for bacteria and yeasts, and 4^7 days for
molds.
 C.-L. Soong et al. / FEMS Microbiology Letters 158 (1998) 51^55 53

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

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54 C.-L. Soong et al. / FEMS Microbiology Letters 158 (1998) 51^55

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

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 C.-L. Soong et al. / FEMS Microbiology Letters 158 (1998) 51^55
imide transformation were cultured in the minimum
liquid medium with succinimide as a sole carbon
source. Several strains listed in Table 1 (bacteria:
Bacillus, Arthrobacter, Pseudomonas; yeasts: Saccha-
romyces; molds: Penicillium, Fusarium) showed sig-
ni¢cant growth. This suggested that cyclic imide-as-
similating activity exists in various microorganisms.
3.3. Successive cyclic imide-transforming enzyme
activities in microorganisms
The successive enzyme activities involved in cyclic
imide transformation were investigated in strains
which showed signi¢cant growth in minimum liquid
medium (Table 1). Succinimide-, succinamic acid-
and succinate-transforming activities were generally
higher in bacteria than in yeasts and molds, and
these activities were expressed at di¡erent levels. In
yeasts and molds, however, the successive transform-
ing activities were nearly equivalent.
4. Discussion
In bacteria, no correlation was observed among
succinimide-, dihydrouracil- and DL-5-methylhydan-
toin-hydrolyzing activities, suggesting that cyclic
imide and cyclic ureide transformation involve di¡er-
ent enzyme systems as observed in Blastobacter sp.
[10]. This may be common in procaryotes. However,
the relative activity of cyclic imide transformation to
that of cyclic ureide transformation was similar in all
fungi examined. Together with the reports that both
cyclic imides and cyclic ureides are hydrolyzed by
mammalian dihydropyrimidinases [8,9], the present
¢ndings suggest that these two activities are cata-
lyzed by an identical enzyme system in eucaryotes.
In summary, we revealed that cyclic imide-trans-
forming activity commonly exists in microorganisms,
FEMSLE 7927 5-1-98
55
and that cyclic imides can be utilized as an energy
source for growth. Since a few natural cyclic imide
compounds are known, the physiological role of cy-
clic imide-transforming activity may be in degrada-
tion of xenobiotics [9]. The present ¢ndings clearly
show that microorganisms can transform cyclic
imides.
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