Báo cáo khoa học: The 3-ureidopropionase of Caenorhabditis elegans, an enzyme involved in pyrimidine degradation pptx

The 3-ureidopropionase of Caenorhabditis elegans ,
an enzyme involved in pyrimidine degradation
Tim Janowitz, Irene Ajonina, Markus Perbandt, Christian Woltersdorf, Patrick Hertel, Eva Liebau
and Ulrike Gigengack
Institut fu
¨
r Zoophysiologie, Westfa
¨
lische Wilhelms-Universita
¨
t, Mu
¨
nster, Germany
Introduction
Pyrimidine nucleotides, besides being constituents of
nucleic acids, fulfil diverse important functions in the
cell. Their cellular concentration is controlled by
de novo synthesis, salvage of preformed molecules and
degradation. The most common route to pyrimidine
degradation is via the reductive pathway [1]. In addi-
tion to this pathway, other routes to pyrimidine degra-
dation exist [2,3]. The reductive route to pyrimidine
degradation consists of three enzymatic steps. First, the
pyrimidine molecule is reduced in a NADPH-dependent
Keywords
b-alanine; biochemical characterization;
GFP fusion protein; nucleotides
Correspondence
T. Janowitz, Institut fu
¨
r Zoophysiologie,

Westfa
¨
lische Wilhelms-Universita
¨
t,
Hindenburgplatz 55, D-48143 Mu
¨
nster,
Germany
Fax: +49 251 8321766
Tel: +49 251 8321710
E-mail:
Note
To prevent confusion and ambiguity, in the
present study, we use the terms ‘3-ureido-
propionase’ instead of b-alanine synthase
and ‘ureido’ to refer to a carbamoylamino-
group. Other common nomenclatures are
given in parenthesis where appropriate
(Received 19 February 2010, revised 23 July
2010, accepted 3 August 2010)
doi:10.1111/j.1742-4658.2010.07805.x
Pyrimidines are important metabolites in all cells. Levels of cellular pyrimi-
dines are controlled by multiple mechanisms, with one of these comprising
the reductive degradation pathway. In the model invertebrate Caenorhabditis
elegans, two of the three enzymes of reductive pyrimidine degradation have
previously been characterized. The enzyme catalysing the final step of
pyrimidine breakdown, 3-ureidopropionase (b-alanine synthase), had only
been identified based on homology. We therefore cloned and functionally
expressed the 3-ureidopropionase of C. elegans as hexahistidine fusion

protein. The purified recombinant enzyme readily converted the two
pyrimidine degradation products: 3-ureidopropionate and 2-methyl-3-urei-
dopropionate. The enzyme showed a broad pH optimum between pH 7.0
and 8.0. Activity was highest at approximately 40 °C, although the half-life
of activity was only 65 s at that temperature. The enzyme showed clear
Michaelis–Menten kinetics, with a K
m
of 147 ± 26 lm and a V
max
of
1.1 ± 0.1 UÆmg protein
)1
. The quaternary structure of the recombinant
enzyme was shown to correspond to a dodecamer by ‘blue native’ gel elec-
trophoresis and gel filtration. The organ specific and subcellular localiza-
tion of the enzyme was determined using a translational fusion to green
fluorescent protein and high expression was observed in striated muscle
cells. With the characterization of the 3-ureidopropionase, the reductive
pyrimidine degradation pathway in C. elegans has been functionally char-
acterized.
Structured digital abstract
l
MINT-7986015: 3-ureidopropionase (uniprotkb:Q19437) and 3-ureidopropionase (uniprotkb:
Q19437) bind (MI:0407)byblue native page (MI:0276)
Abbreviations
GFP, green fluorescent protein; RNAi, RNA interference.
4100 FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS
manner by dihydropyrimidine dehydrogenase (EC 1.3.
1.2) with a subsequent ring opening by dihydro-
pyrimidinase (EC 3.5.2.2). In the last step, the formed

ureido compound is hydrolyzed by 3-ureidopropionase
(b-alanine synthase, N-carbamoyl-b-alanine amidohy-
drolase; EC 3.5.1.6) to carbon dioxide, ammonia and a
b-amino acid (Fig. S1). Almost all known 3-ureidopro-
pionases, excluding yeast 3-ureidopropionase, belong to
branch 5 of the so-called nitrilase superfamily of
enzymes. Members of this superfamily all possess a
conserved cysteine residue that is essential for enzy-
matic activity [4–6]. Therefore, the addition of low
amounts of reducing agents such as dithiothreitol have
been reported to result in increased activity, presumably
as a result of stabilization of the reduced state of the
catalytic residue [7–9]. For 3-ureidopropionases purified
from rat and maize, a dependence of enzymatic activity
on Zn
2+
-ions has been reported [9,10].
The enzymes involved in pyrimidine catabolism uti-
lize both uracil and thymine as substrates. Cytosine is
not directly accepted as a substrate and must first be
deaminated to uracil. The b-amino acid resulting from
degradation can be channelled into energy metabolism
via a semi-aldehyde intermediate [11]. A fraction of the
resulting b-amino acid can also fulfil other functions in
the cell. The degradation product of uracil, 3-amino-
propionate (b-alanine), for example, can be condensed
with histidine to form the dipeptide carnosine. Carno-
sine and other similar dipeptides containing nonprotein-
ogenic amino acids can be found in excitable tissues,
brain and skeletal muscles. The physiological role of

such dipeptides is not yet understood [12]. Defects in
pyrimidine degradation are known to be the cause of
several human disorders [13–15]. The clinical symp-
toms of patients suffering form such disorders are very
diverse, ranging from asymptomatic to severe symp-
toms, with mental retardation and convulsive attacks
being the most common. Even a normally asymptom-
atic partial deficiency of dihydropyrimidine dehydro-
genase can cause severe complications for patients
receiving chemotherapy with pyrimidine analogs such
as fluorouracil, as a result of a diminishing of the nor-
mally high turnover rates and subsequent overdosing
[16,17].
In the genome of the nematode Caenorhabditis ele-
gans, only the reductive pathway (and none of the
alternative routes) is present. The first two enzymes of
reductive pyrimidine degradation in C. elegans, dihy-
dropyrimidine dehydrogenase [18] and dihydropyrimi-
dinase [19], have already been characterized by genetic
and ⁄ or molecular methods. The enzyme catalyzing the
last step, 3-ureidopropionase, has so far only been
identified based on homology to 3-ureidopropionases
of other organisms [4]. Because such predictions can
be misleading [8], it is important to verify them at a
functional level. Accordingly, we cloned the cDNA
coding for the predicted 3-ureidopropionase of C. ele-
gans and functionally expressed it as a hexahistidine
fusion protein in Escherichia coli. The purified recom-
binant protein was then functionally characterized
in vitro. To analyze the expression pattern of the 3-ure-

idopropionase, transgenic C. elegans were created
expressing a green fluorescent protein (GFP) fusion
protein under the control of the 3-ureidopropionase
promoter.
Results and Discussion
Cloning and expression of recombinant
3-ureidopropionase
The sole homolog of 3-ureidopropionase in C. elegans
is encoded by the gene F13H8.7. The protein encoded
by this gene clusters together with other 3-ureidopropi-
onases of this family during phylogenetic analysis
(Fig. 1). The 3-ureidopropionases used for phyloge-
netic inferrence, similar to almost all 3-ureidopropion-
ases identified so far, belong to the nitrilase
superfamily of enzymes [4]. An exception is the enzyme
from Saccharomyces (Lachancea) kluyveri. This enzyme
appears to be phylogenetically unrelated to 3-ureido-
propionases of other eukaryotes because it shows high
structural similarity with dizinc-dependent exopepti-
dases [20,21]. It has been proposed that this enzyme is
prototypic of fungal 3-ureidopropionases. A homology
model (Fig. S2) of the C. elegans enzyme based on the
closely-related 3-ureidopropionase of Drosophila mela-
nogaster (Dme3-UP; Fig. 1) was constructed. The cata-
lytic triade Glu-Lys-Cys typical for enzymes of the
nitrilase superfamily [4] could be observed in the
model. For functional characterization, the cDNA of
gene F13H8.7 was expressed as hexahistidine fusion
protein in E. coli. The purified enzyme liberated
ammonia upon incubation with 3-ureidopropionate

(not shown). To verify that 3-aminopropionate (b-ala-
nine) was produced, a sample from an activity assay
was analyzed by MS. We were able to identify two
substances with m ⁄ z ratios corresponding to 3-amino-
propionate and 3-ureidopropionate. Furthermore, the
MS ⁄ MS spectra of both ions corresponded to the
MS ⁄ MS spectra of genuine 3-aminopropionate and
3-ureidopropionate respectively (data not shown). We
therefore conclude that the recombinant protein shows
a genuine 3-ureidopropinase activity, confirming that
the gene product of F13H8.7 is the C. elegans 3-urei-
dopropionase.
T. Janowitz et al. 3-Ureidopropionase of C. elegans
FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS 4101
Biochemical characterization of recombinant
3-ureidopropionase
On the basis of bioinformatics and the homology
model (Fig. S2), a catalytically relevant cysteine was
shown to be present in the C. elegans 3-ureidopropion-
ase. To prevent oxidation of this residue common to
all members of the nitrilase superfamily, low millimo-
lar amounts of dithiothreitol were added to the activity
assays. The activity of the enzyme was higher with
0.25 mm dithiothreitol added than without dithiothrei-
tol or with higher concentrations. The enzyme did not
show any dependence of activity on Zn
2+
-ions, which
had been reported for 3-ureidopropionases from other
species [9,10]. Incubation with either 1 mm EDTA or

1mm ZnCl
2
did not result in any change in activity
compared to control reactions without additive. A
reaction mechanism independent of divalent cations
has been theoretically deduced for carbamylases, repre-
senting another branch of ureido-group hydrolyzing
enzymes [22]. Given that 3-ureidopropionases are clo-
sely related to carbamylases (Fig. 1) and a recombi-
nant 3-ureidopropionase from D. melanogaster also
does not show any effect of Zn
2+
ions on enzymatic
activity [23], the dependence of 3-ureidopropionase
activity on Zn
2+
ions appears to be a peculiarity of
some species. To test the substrate specificity of the
recombinant 3-ureidopopionase, several ureido-com-
pounds with different side chain architectures and also
compounds with functional groups similar to the ureido-
group (e.g. guanidino-group) were tested in activity
assays. Besides uracil-derived 3-ureidopropionate, the
recombinant enzyme also accepted thymine-derived
2-methyl-3-ureidopropionate (relative activity com-
pared to 3-ureidopropionate: 82 ± 18%). 4-Amino-4-
oxo-butanoic acid, which also appeared to show some
conversion, proved not to be a reliable substrate;
therefore, this result is not discussed further. Other
substances tested were no substrate of the C. elegans

3-ureidopropionase (Table 1). The recombinant C. ele-
gans enzyme did not show any activity with ureido-
acetic acid and 2-ureidopropionic acid, as reported
previously for the enzyme purified from rat liver [24].
Because the recombinant enzyme showed highest activ-
ity with 3-ureidopropionate, this compound was used
in all further activity measurements.
The pH optimum of the enzymatic activity was
determined to be quite broad, with an pH optimum
between pH 7.0 and 8.0. The enzyme showed maximal
activity at approximately 40 °C(Fig. 2). However, at
this temperature, the C. elegans enzyme proved to be
unstable. After preincubation at 40 °C and activity
measurement at 30 °C (where activity was stable for
‡ 2 h), the half-life of enzymatic activity was only 65 s.
The activity of the enzyme remained stable at a specific
activity of 0.2 UÆmg protein
)1
after 5 min of preincu-
bation. The dependence of the activity on substrate
concentration showed clear Michaelis–Menten kinetics.
Nonlinear regression of the experimental data to
the Michaelis–Menten equation yielded a K
m
of
147 ± 26 lm and a V
max
of 1.1 ± 0.1 UÆmg protein
)1
(Fig. 3). In rat and humans, 3-ureidopropionase has

Branch 3
Branch 5
Branch 11
Branch 6
Branch 1
Branch 2
Branch 10
Branch 8
Branch 7
Branch 9
Branch 4
Bootstrap support
0–50
50–90
90–100
0.1 changes/site
Fig. 1. Phylogenetic tree of the nitrilase
superfamily. For tree construction, protein
sequences of known members of the
nitrilase superfamily were aligned using
CLUSTALX. For phylogenetic inference, the
PHYML maximum likelihood method was
used with 100 bootstrap trials. Definitions of
the different branches and accession num-
bers of proteins used are given in Table S1.
Saccharomyces kluyveri 3-ureidopropionase
has been excluded from the analysis
because it belongs to a different phylo-
genetic group.
3-Ureidopropionase of C. elegans T. Janowitz et al.

4102 FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS
been reported to show positive co-operativity with
3-ureidopropionate as substrate [25,26]. Such kinetic
behaviour is best described by the Hill equation. A
nonlinear regression of our data to the Hill equation
showed that the data fitted best for n  1 (when the
Hill equation transforms into the Michaelis–Menten
equation) (not shown). Other 3-ureidopropionases
characterized so far from plants and animals all show
a lower K
m
value (Table 2) than the C. elegans
enzyme. Only the enzyme from S. kluyveri, which
belongs to a different phylogenetic group, has an
approximately 500-fold higher K
m
. Differences in the
reaction mechanism as a result of the large phyloge-
netic distance might be responsible for such a discrep-
ancy. In plants, the 3-ureidopropionase is involved in
the synthesis of the pantothenate moity of coenzyme A
and 3-aminopropionate can serve as osmoprotectant
[9]. Those different roles in metabolism might be
responsible for the observed differences in K
m
values
of 3-ureidopropionase of C. elegans and plants. It is
quite unexpected that the K
m
of the D. melanogaster

enzyme is approximately six-fold lower because both
enzymes show a high degree of sequence identity. Fur-
thermore, the homology model of the C. elegans
enzyme corresponds well with the D. melanogaster
template. To explain the difference in K
m
values, it
might be valuable to solve the actual structure of the
C. elegans enzyme.
Determination of native protein mass
To determine the native molecular mass of the recom-
binant C. elegans 3-ureidopropionase, gel filtration
Table 1. Substrate specificity of recombinant 3-ureidopropionase from Caenorhabditis elegans. Enzymatic activity was measured by quanti-
fying the amount of ammonia released during reactions. Substrates were provided at a concentration of 3–10 m
M. 100% activity equals
0.88 ± 0.10 UÆmg protein
)1
. ND, not detectable.
Substrate Activity (%) Linear formula
3-Ureidopropionic acid (N-carbamyl-b-alanine) 100 COOH(CH
2
)
2
NHCONH
2
2-Methyl-3-ureidopropionic acid (N-Carbamyl-b-aminoisobutyric acid) 82 ± 18
a
COOH(CHCH
3
)CH

2
NHCONH
2
4-Amino-4-oxo-butyric acid
b
(succinamic acid) 3 ± 1 COOH(CH
2
)
2
CONH
2
2-Ureidoacetic acid (N-carbamylglycine) ND COOHCH
2
NHCONH
2
4-Ureidobutanoic acid (N-carbamyl-c-aminobutyric acid) ND
a
COOH(CH
2
)
3
NHCONH
2
2-Ureidopropionic acid (N-carbamy-a-alanine) ND
a
COOH(CHNHCONH
2
)CH
3
2-Ureidobutanedioic acid (N-carbamylaspartic acid) ND COOH(CHNHCONH

2
)CH
2
COOH
3-Guanidinopropionic acid ND COOH(CH
2
)
2
NHCNHNH
2
(S)-2-Amino-5-ureidopentanoic acid (L-citrulline) ND COOH(CHNH
2
)(CH
2
)
3
NHCONH
2
1-Ureido-4-aminobutane (N-carbamylputrescine) ND NH
2
(CH
2
)
4
NHCONH
2
N-Allylurea
b
ND CH
2

CHCH
2
NHCONH
2
a
The substance was synthesized and crude synthesis solution used for the experiments.
b
The substance was not stable under the experi-
mental conditions and influenced the ammonia determination.
–0.2
0
–5 0 5 10 15 20 25 30 35 40 45 50 55 60 65
Temperature (°C)
Specific activity (U·mg protein
–1
)
0.2
0.4
0.6
0.8
1
1.2
Fig. 2. Temperature dependence of the 3-ureidopropionase reac-
tion. The enzyme showed maximal activity at approximately 40 °C.
0
0
0.2
0.4
0.6
0.8

1
1.2
1.4
500 1000 1500 2000
Substrate concentration (µ
M)
Specific activity (U·mg protein
–1
)
2500 3000 3500
Fig. 3. Dependence of specific activity on substrate concentration.
Activity was measured with the indophenol blue method using 3-
ureidopropionate as substrate. Data represent the means ± SD of
n ‡ 3 experiments. Data points were used for nonlinear regression
to the Michaelis–Menten equation, resulting in the solid curve
shown.
T. Janowitz et al. 3-Ureidopropionase of C. elegans
FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS 4103
separation and ‘blue native’ PAGE was used. Here,
the apparent molecular mass was 472 ± 4 kDa, which,
taking a monomer mass of 43.2 kDa into account,
points to a dodecamer (Fig. 4). To support this find-
ing, the oligomeric state of the enzyme, the recombi-
nant protein was subjected to gel filtration using a
calibrated Superdex S-200 column, followed by
immunodetection of the His-tagged protein. The
protein eluted as a single peak corresponding to a
molecular mass of approximately 500 kDa (data not
shown). Because temperatures of 40 °C had an influ-
ence on activity (vide supra), we also preincubated

samples at 40 °C before electrophoresis. The resulting
protein pattern showed an additional signal at
404 ± 5 kDa (Fig. 4). Taking into consideration the
decreased activity of enzyme that was preincubated at
40 °C, it can be speculated that the 404 kDa oligomer
represents an inactive enzyme species diminished of its
overall activity. 3-Ureidopropionases purified from rat
also have been reported to have different enzymatic
activities, depending on their oligomerization states.
However, in these cases, the changes in oligomerization
state were ligand-dependent [26]. We could not observe
a similar behaviour for the C. elegans enzyme (data
not shown). The recombinant D. melanogaster enzyme
also does not to show ligand-dependent oligomeriza-
tion [23]. Conclusive data on whether this is of any
significance for the regulation of 3-ureidopropionase in
different phylogenetic groups is presently unavailable.
Characterization of 3-ureidopropionase in vivo
The physiological relevance of 3-ureidopropionase for
C. elegans is only poorly characterized. In one large-
scale RNA interference (RNAi) experiment, maternal
sterility has been reported [27], although this effect was
not found in two other experiments [28,29]. When
exposing worms to RNAi, we were also unable to
observe any phenotypic abnormalities compared to
wild-type animals. As described, low doses of 5-fluoro-
uracil (5-FU) completely block F1 embryo hatching in
wild-type worms [30]. To determine whether 3-ureido-
propionase deficiency has any effect on worms exposed
to 5-FU, RNAi- and wild-type worms were incubated

with low doses of 5-FU. No significant difference was
observed in the number of hatched F
1
embryos
(Fig. S3). This is also observed in the D. melanogaster
pyd mutants (loss-of function mutation of 3-ureidopro-
pionase) that were fed dietary 5-FU. Whereas su(r)
mutants (loss-of-function mutation of dihydropyrimi-
dine dehydrogenase) are hypersensitive to 5-FU as a
result of its accumulation, this is reduced in CRMP
mutants (loss-of-function mutation dihydropyrimidinase)
Table 2. Comparison of kinetic parameters of eucaryotic 3-ureidopropionases. V
max
and either K
m
or K
½
are shown depending on the cata-
lytic mechanism. Only data reporting 3-ureidopropionate as substrate are included. Amino acid identities (aa identity) are given with refer-
ence to the sequence of C. elegans 3-ureidopropionase. h, Hill coefficent; ND, not determined.
Organism
V
max
(UÆmg protein
)1
) K
m
(lM) K
½
(lM) h

Amino acid
identity (%) Purification Reference
C. elegans 1.1 147 – – 100 Recombinant Present study
Drosophila melanogaster 0.46 24.5 – – 66 Recombinant [21]
Zea mays ND 11 – – 63 Partial [9]
Arabodpsis thaliana 0.47 6 – – 64 Recombinant [21]
Rat ND – 170 2.0 64 1045-fold [24]
Rat
a
ND 8 ND 1.9 64 1096-fold [26]
Homo sapiens ND – 100 2.0 65 Recombinant [25]
Euglena gracilis ND 38 – – –
b
95.5-fold [45]
Saccharomyces kluyveri
c
7.3 70900 – – 7 Recombinant [21]
a
Co-operativity only below 12 nM of substrate.
b
No sequence information is available.
c
Belongs to a different phylogenetic group.
NH
480
720
242
M
kDa
Fig. 4. ‘Blue native’ gel electrophoresis of recombinant protein. In

lane N, 2 lg of native protein were loaded onto the gel. In lane H,
5 lg of protein were incubated for 5 min at 40 °C before loading.
Separation was performed on a 4–14% (w ⁄ v) polyacrylamide gradi-
ent. Lane M shows the native protein standard.
3-Ureidopropionase of C. elegans T. Janowitz et al.
4104 FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS
making them less sensitive to 5-FU. Because little
or no 5-FU accumulates in the pyd mutants, they
exibit essentially wild-type sensitivity towards the drug
[31].
Transgenic worms were created that expressed a
3-ureidopropionase-GFP fusion protein under the con-
trol of the 3-ureidopropionase promoter. GFP signals
were detected during all stages of C. elegans develop-
ment (larvae L1–4 and adult hermaphrodites). Strong
GFP expression was observed in dense bodies of stri-
ated body wall muscle cells (Fig. 5). C. elegans has
striated and nonstriated muscles. The nonstriated mus-
cles are the pharyngeal, intestinal, uterine, vulval and
anal muscles, whereas the body wall muscles are stri-
ated. In C. elegans, sarcomere attachment to the mus-
cle membrane and the underlying basement membrane
is performed by the dense body. This protein complex
shares functional similarity with both the vertebrate
Z-disk and the costamere. In addition to its structural
role, the dense body also performs a signalling func-
tion in muscle cells and communication between dense
bodies and nuclei has been postulated [32]. However,
colocalization experiments using 4¢,6¢-diamidino-2-
phenylindole- and Hoechst staining indicated that

3-ureidopropionase is not localized in the nuclei of
muscle cells (data not shown). In animals, catabolic
processes are often associated with ‘liver-like’ organs.
In humans, 3-ureidopropionase is expressed in the
liver. In rat, it has been purified and cloned from liver
tissue [10,33]. In C. elegans, the tissue that performs
‘liver-like’ function is the intestine. Therefore, the
absence of 3-ureidopropionase expression is unex-
pected. Presuming that there really is no expression of
3-ureidopropionase in the C. elegans intestine, this
might indicate that its primary function does not lie in
the degradation of pyrimidines. The localization in
striated muscle cells might rather point to a role in
providing b-amino acids for synthesis of dipeptides
such as carnosine. Reporter gene analyses of dihydro-
pyrimidinase in C. elegans also demonstrated expres-
sion in body wall muscle cells [19]. Currently, the
localization of dihydropyrimidine dehydrogenase has
not been investigated. Therefore, at least two of the
three enzymes of reductive pyrimidine degradation are
known to occur in striated body wall muscle cells.
Further studies will continue to investigate the func-
tional role of reductive pyrimidine degradation and
synthesis of carnosin-like dipeptides in striated muscle
cells of C. elegans.
Experimental procedures
Organisms and growth conditions
Caenorhabditis elegans strain N2 (Bristol variety) was used in
the present study. Unless noted otherwise, nematodes were
grown at 20 °C on nematode growth medium with E. coli

OP50 provided as a food source ad libitum [34]. E. coli was cul-
tivated in appropriate media under selection pressure at 37 °C.
General molecular biological procedures
Total RNA was prepared using TRIreagent (Segenetic,
Borken, Germany) and checked for integrity by denaturing
agarose gel electrophoresis. The first strand cDNA synthesis
kit (Fermentas, St. Leon-Rot, Germany) was used for
subsequent cDNA synthesis. Dephosphorylation of vector
DNA was performed with calf intestine alkaline phospha-
A
B
C
Fig. 5. Analysis of the expression pattern
of 3-ureidopropionase in Caenorhabditis
elegans. GFP images of adult C. elegans
carrying 3-ureidopropionase::GFP are
shown. (A) Confocal differential interference
contrast micrograph, (B) combined confocal
differential interference contrast and
fluorescence micrograph (C) fluorescence
micrograph. Scale bar = 10 lm.
T. Janowitz et al. 3-Ureidopropionase of C. elegans
FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS 4105
tase (Promega, Mannheim, Germany) in accordance with
the manufacturer’s instructions.
Cloning of 3-ureidopropionase
The cDNA of 3-ureidopropionase (gene F13H8.7) was
amplified from a cDNA library made of 1 lg of total RNA
using primers 3-UP_F (5¢-CATATGTCTGCAGCTCCG
GCT-3¢) and 3-UP_R (5¢-CTCGAGTTGCTCTCTTCT

GATGTCTG-3¢). For amplification of promoter fragments,
genomic DNA was used as a template with primers
Prom.UP_F (5¢-AAGCTTAAGTCAATGTGGGCAAG-3¢)
and Prom.UP_R (5¢-CATATGTTTTACCTGAATAAGAT-
A-3¢). Primers used in the PCRs introduced restriction sites
that were used for subsequent cloning. PCR-fragments were
cloned into the pJET2.1 ⁄ blunt vector (Fermentas). Errors
introduced during PCR were excluded by sequencing. For
recombinant expression, cDNA was introduced NdeI ⁄ XhoI
into pET22b(+) (excising the pelB leader sequence). To
make translational GFP fusions, the 3-ureidopropionase
cDNA was inserted NdeI ⁄ SalI into a pJET plasmid contain-
ing a promoter fragment. The whole construct was excised
with XhoI and then transferred into the SalI site of dephos-
phorylated pPD95.77 (Addgene plasmid 1495). Correct ori-
entation of the insert was checked by restriction analysis.
Purification of heterologously expressed proteins
Bacterial expression of recombinant protein was performed
in E. coli BL21-CodonPlus
Ò
(DE3)-RIL cells (Stratagene,
La Jolla, CA, USA). Expression cultures of 300 mL
2YT medium (16 gÆL
)1
tryptone, 10 gÆL
)1
yeast extract,
5gÆL
)1
NaCl, pH 7.0–7.5) supplemented with ampicillin

were inoculated at a dilution of 1 : 10 from a saturated over-
night culture. After 1 h of incubation at 25 °C and agitation
by rotary shaking at 170 r.p.m., expression was induced by
addition of 1 mm isopropyl thio-b-d-galactoside. Cultures
were subsequently incubated for 6 h at 25 °C with
agitation by rotary shaking at 170 r.p.m. Cells were har-
vested by centrifugation and resuspended into lysis buffer
(50 mm NaH
2
PO
4
, pH 8.0, 300 mm NaCl) supplemented
with 5 mm 2-mercaptoethanol, 0.6 gÆL
)1
lysozyme. Cell lysis
was performed by sonification (five bursts of 1 min) after
30 min of incubation on ice. Cell debris was removed by
centrifugation. The crude protein supernatant was passed
over a Ni
2+
-NTA-agarose column (Qiagen, Hilden, Ger-
many; 2 mL matrix) equilibrated in lysis buffer with 10 mm
imidazole. The column was washed with approximately ten
volumes of lysis buffer with 30 mm imidazole. Bound pro-
teins were eluted with 2.5 mL of lysis buffer with 250 mm
imidazole and desalted over a PD10 column (GE Health-
care, Mu
¨
nchen, Germany). Desalted protein was stored in
50 mm K-phosphate (pH 8.0), 1 mm dithiothreitol at

)80 °C. The purity of the recombinat protein was ‡ 85% as
judged by SDS ⁄ PAGE.
Molecular size of the 3-ureidopropionase
The molecular size and oligomeric state of the 3-ureidopro-
pionase was assessed by subjecting the affinity-purified pro-
tein to FPLC on a Superdex S-200 column (Amersham
Biosciences, Piscataway, NJ, USA). The Superdex S-200
column, equilibrated with 100 mm sodium phosphate (pH
7.0) was calibrated using a gelfiltration standard (151–1901;
Bio-Rad, Munich, Germany) containing thyroglobulin,
a-globulin, ovalbumin, myoglobin and vitamin B
12
. ‘Blue
native’ electrophoresis was performed as described previ-
ously [35], using a 4–14% (w ⁄ v) polyacrylamide gradient
and separating 2–5 lg of protein.
Determination of enzymatic activity
A standard reaction for determination of enzymatic activities
was performed with 3.5 lgÆmL
)1
protein in 100 mm K-phos-
phate (pH 7.5), 0.25 mm dithiothreitol at 3 °C and substrates
provided at a final concentration of 3–10 mm. Enzymatic
activity was determined by measuring ammonia liberated
during reactions with the indophenol blue method [36,37]. In
brief, 100 lL of sample were mixed with 100 lL each of
0.33 m sodium phenolate, 0.02 m sodium hypochlorite and
0.01% (w ⁄ v) sodium pentacyanonitrosylferrate. After
incubation at approximately 95 ° C for 2 min, samples were
diluted with 600 lL of water and A

640
was measured.
Ammonia concentrations were assessed using standard
curves contructed with ammonium chloride. Background
values of samples with inactive protein were subtracted.
Chemical synthesis of ureido compounds
Chemical synthesis of ureido compounds as substrates for
activity assays was carried out as described previously [38].
Yield of synthesis was determined by TLC on a SIL ⁄ G
matrix developed in chloroform ⁄ methanol ⁄ formic acid
(65 : 18 : 1) [39]. Amines were stained by spraying with nin-
hydrin [0.2% (w ⁄ v) in ethanol] and subsequent incubation at
80 °C. Ureido compounds were visualized by spraying with
4-(dimethylamino)-benzaldehyde [1% (w ⁄ v) in hydrochloric
acid ⁄ methanol (1 : 1)]. Synthesis yield as judged by TLC was
‡ 80%. Synthesized substrates (2-methyl-3-ureidopropionic
acid, 4-ureidobutyric acid and 2-ureidopropionic acid) were
directly used for activity measurements without further puri-
fication. Synthesized 3-ureidopropionic acid showed almost
identical behaviour in activity measurements as the commer-
cially available (Sigma, Steinheim, Germany) compound.
MS determination of reaction products
A standard reaction was performed as described above,
except that 5 lgÆmL
)1
enzyme were used in 5 m m K-phos-
phate (pH 8.0). Samples were prepared for MS as described
3-Ureidopropionase of C. elegans T. Janowitz et al.
4106 FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS
previously [8], with the modification that dry samples were

resuspended in 1% (v ⁄ v) formic acid in methanol ⁄ water
(1 : 1). MS was performed by Simone Ko
¨
nig of the core unit
‘Integrated Functional Genomics’ of the Interdisciplinary
Center for Clinical Research Mu
¨
nster (Germany). Manual
nanospray MS ⁄ MS using a modified stage [40] was carried
out with Q-TOF Premier (Waters Corp., Manchester, UK).
RNAi
For RNAi experiments, double-stranded RNA was pro-
duced in the E. coli strain HT115 transformed with the
L4440 feeding vector pPD129.36 (L4440) that contained a
cDNA fragment of the 3-ureidopropionase. Isopropyl thio-
b-d-galactoside (1 mm) was added to induce transcription
of the double-stranded RNA. To determine whether 3-urei-
dopropionase deficiency has any effect on worms exposed
to 5-FU, RNAi worms, as well as worms feeding on E. coli
harbouring the L4440 vector only, were incubated with low
doses of 5-FU. Because concentrations above 0.2 lgÆmL
)1
resulted in a severe egg laying defect, 0.1 and 0.05 lgÆmL
)1
were chosen. Progenies of 18 worms (3 · 6 plates) were
counted and the experiments were performed twice.
Transformation of worms using microinjection
and fluorescence microscopy of GFP fusion
proteins
Transgenic C. elegans germline transformation was per-

formed by coinjecting the vector construct 3-ureidopropion-
ase::GFP with the pRF4 plasmid encoding the dominant
marker gene rol-6 into the germline of young adults
(Fig. 5A). To investigate the cell-specific, developmentally
regulated transcription of 3-ureidopropionase, GFP expres-
sion patterns were analyzed by fluorescence microscopy.
Images were captured with a Zeiss axiovert 100 microscope
(Carl Zeiss, Oberkochen, Germany) equipped with fluores-
cein isothiocyanate ⁄ GFP filters. Hoechst-staining of nuclei
was performed as described previously [41].
Construction of a phylogenetic tree and
structural model
Full length protein sequences were extracted from the
NCBI protein database ().
Sequences were trimmed to the same length and aligned
using clustalx [42]. Alignment parameters were optimized
until at least the three catalytic residues (Glu, Lys, Cys) [4]
were aligned correctly. This data set was used for phyloge-
netic inference with the phyml online platform [43] with
100 bootstrap trials. The resulting tree was visualized using
treeview, version 1.6.6 ( />rod/treeview.html). Accession numbers of proteins used in
phylogenetic analysis are presented in Table S1.
For illustrative purposes, a 3D model was generated
based on the crystal structure of the D. melanogaster 3-urei-
dopropionase (Protein Data Bank code: 2VHH) (Fig. S2).
The swiss-model workspace [44] was used with standard
settings, and molecular visualization was conducted using
pymol (The PyMOL Molecular Graphics System, version
1.2r3pre; Schro
¨

dinger, LLC, Mannheim, Germany).
Acknowledgements
Some of the organisms used in the present study were
kindly provided by the Caenorhabditis Genetics Center
(funded by the NIH National Center for Research
Resources). Andrew Fire is acknowledged for the plas-
mid pPD95.77. Some of the substrates tested were
donated by Markus Piotrowski. Financial and material
support for this project was generously provided by
Ru
¨
diger J. Paul.
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Supporting information
The following supplementary material is available:
Fig. S1. Metabolism of 3-aminopropionate.
Fig. S2. Homology model of the subunit of 3-ureido-
propionase, based upon the crystal structure of the
D. melanogaster 3-ureidopropionase (Protein Data
Bank code: 2VHH).

Fig. S3. To determine whether 3-ureidopropionase
deficiency has any effect on worms exposed to 5-FU,
RNAi- and control worms (carrying the feeding vector
L4440) were incubated with low doses of 5-FU.
Table S1. Proteins used in phylogenetic analysis.
Enzyme classes and branches were assigned as
described previously [4].
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
T. Janowitz et al. 3-Ureidopropionase of C. elegans
FEBS Journal 277 (2010) 4100–4109 ª 2010 The Authors Journal compilation ª 2010 FEBS 4109