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

First, the pyrimidine molecule is reduced in a NADPH-dependent Keywords b-alanine; biochemical characterization; GFP fusion protein; nucleotides Correspondence T.. In the model invertebr

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: tim.janowitz@rub.de

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 40C, although the half-life

of activity was only 65 s at that temperature The enzyme showed clear Michaelis–Menten kinetics, with a Km of 147 ± 26 lm and a Vmax 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 translalocaliza-tional 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

Abbreviations

GFP, green fluorescent protein; RNAi, RNA interference.

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 Zn2+-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-ureido3-amino-propionate 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

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 Zn2+-ions, which

had been reported for 3-ureidopropionases from other

species [9,10] Incubation with either 1 mm EDTA or

1 mm ZnCl2 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 Zn2+ ions on enzymatic

activity [23], the dependence of 3-ureidopropionase

activity on Zn2+ 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-ureido-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 40C and activity measurement at 30C (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 Km of

147 ± 26 lm and a Vmax 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.

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 Km 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 Km 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 Km values

of 3-ureidopropionase of C elegans and plants It is quite unexpected that the Km 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 Km values, it might be valuable to solve the actual structure of the

C elegansenzyme

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.

2-Methyl-3-ureidopropionic acid (N-Carbamyl-b-aminoisobutyric acid) 82 ± 18 a COOH(CHCH3)CH2NHCONH2

a

The substance was synthesized and crude synthesis solution used for the experiments.bThe 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)

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 )

–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.

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 40C had an

influ-ence on activity (vide supra), we also preincubated

samples at 40C 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

40C, 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 F1 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 Vmax and either Km or K½ 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 sequrefer-ence of C elegans 3-ureidopropionase h, Hill coefficent; ND, not determined.

Organism

Vmax (UÆmg protein)1) Km(l M ) K½(l M ) h

Amino acid identity (%) Purification Reference

a Co-operativity only below 12 n M of substrate b No sequence information is available c Belongs to a different phylogenetic group.

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.

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 communicafunc-tion 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 elegansstrain N2 (Bristol variety) was used in the present study Unless noted otherwise, nematodes were grown at 20C 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 37C

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.

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 (DE3)-RIL cells (Stratagene,

La Jolla, CA, USA) Expression cultures of 300 mL

2YT medium (16 gÆL)1 tryptone, 10 gÆL)1 yeast extract,

5 gÆL)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 25C 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 25C with

agitation by rotary shaking at 170 r.p.m Cells were

har-vested by centrifugation and resuspended into lysis buffer

(50 mm NaH2PO4, pH 8.0, 300 mm NaCl) supplemented

with 5 mm 2-mercaptoethanol, 0.6 gÆL)1lysozyme 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 Ni2+-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-ureidopionase 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 B12 ‘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)1protein in 100 mm K-phos-phate (pH 7.5), 0.25 mm dithiothreitol at 3C 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 95C for 2 min, samples were diluted with 600 lL of water and A640 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

80C 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 mm K-phos-phate (pH 8.0) Samples were prepared for MS as described

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 (http://www.ncbi.nlm.nih.gov)

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 (http://taxonomy.zoology.gla.ac.uk/

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

References

1 Wasternack C (1980) Degradation of pyrimidines and pyrimidine analogs-pathways and mutual influences Pharmacol Ther 8, 629–651

2 Hayaishi O & Kronberg A (1952) Metabolism of cyto-sine, thymine, uracil, and barbituric acid by bacterial enzymes J Biol Chem 197, 717–732

3 Andersen G, Bjo¨rnberg O, Polakova S, Pynyaha Y, Rasmussen A, Møller K, Hofer A, Moritz T, Sandrini MPB, Merico A-M et al (2008) A second pathway to degrade pyrimidine nucleic acid precursors in eukary-otes J Mol Biol 380, 656–666

4 Pace HC & Brenner C (2001) The nitrilase superfamily: classification, structure and function Genome Biol 2, reviews0001.1–reviews0001.9

5 Brenner C (2002) Catalysis in the nitrilase superfamily Curr Opin Struct Biol 12, 775–782

6 Bork P & Koonin EV (1994) A new family of carbon-nitrogen hydrolases Protein Sci 3, 1344–1346

7 Vorwerk S, Biernacki S, Hillebrand H, Janzik I, Mu¨ller

A, Weiler EW & Piotrowski M (2001) Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2⁄ NIT1 ⁄ NIT3-gene cluster Planta 212, 508–516

8 Piotrowski M, Janowitz T & Kneifel H (2003) Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis J Biol Chem 278, 1708–1712

9 Walsh TA, Green SB, Larrinua IM & Schmitzer PR (2001) Characterization of plant b-ureidopropionase and functional overexpression in Escherichia coli Plant Physiol 125, 1001–1011

10 Kvalnes-Krick KL & Traut TW (1993) Cloning, sequencing, and expression of a cDNA encoding

b-alanine synthase from rat liver J Biol Chem 268,

5686–5693

11 Griffith OW (1986) b-Amino acids: mammalian

metab-olism and utility as a-amino acid analogues Annu Rev

Biochem 55, 855–878

12 Bauer K (2005) Carnosine and homocarnosine, the

for-gotten, enigmatic peptides of the brain Neurochem Res

30, 1339–1345

13 van Gennip AH, Abeling NGGM, Vreken P & van

Kuilenburg ABP (1997) Inborn errors of pyrimidine

degradation: clinical, biochemical and molecular

aspects J Inherit Metab Dis 20, 203–213

14 Nyhan WL (2005) Disorders of purine and pyrimidine

metabolism Mol Genet Metab 86, 25–33

15 Kuilenburg ABP, Meinsma R, Beke E, Assmann B,

Ribes A, Lorente I, Busch R, Mayatepek E, Abeling

NGGM, van Cruchten A et al (2004)

b-Ureidopropion-ase deficiency: an inborn error of pyrimidine

degrada-tion associated with neurological abnormalities Hum

Mol Genet 13, 2793–2801

16 Heggie G, Sommadossi J, Cros D, Huster W & Diasio

R (1987) Clinical pharmacokinetics of 5-fluorouracil in

plasma, urine, and bile Cancer Res 47, 2203–2206

17 van Kuilenburg ABP (2004) Dihydropyrimidine

dehy-drogenase and the efficacy and toxicity of

5-fluoroura-cil Eur J Cancer 40, 939–950

18 Kim S, Park D-H & Shim J (2008) Thymidylate

syn-thase and dihydropyrimidine dehydrogenase levels are

associated with response to 5-fluorouracil in

Caenor-habditis elegans Mol Cells 26, 344–349

19 Takemoto T, Sasaki Y, Hamajima N, Goshima Y,

Nanaka M & Kimura H (2000) Cloning and

character-ization of the Caenorhabditis elegans CeCRMP⁄ DHP-1

and -2; common ancestors of CRMP and

dihydro-pyrimidinase Gene 261, 259–267

20 Beck H, Dobritzsch D & Pisˇkur J (2008)

Saccharo-myces kluyverias a model organism to study pyrimidine

degradation FEMS Yeast Res 8, 1209–1213

21 Andersen B, Lundgren S, Dobritzsch D & Pisˇkur J

(2008) A recruited protease is involved in catabolism of

pyrimidines J Mol Biol, 379, 243–250

22 Nakai T, Hasegawa T, Yamashita E, Yamamoto M,

Kumasaka T, Ueki T, Nanba H, Ikenaka Y, Takahashi

S, Sato M et al (2000) Crystal structure of

N-carbamyl-D-amino acid amidohydrolase with a novel

catalytic framework common to amidohydrolases

Structure 8, 729–737

23 Lundgren S, Lohkamp B, Andersen B, Pisˇkur J &

Dobritzsch D (2008) The crystal structure of b-alanine

synthase from Drosophila melanogaster reveals a

homo-octameric helical turn-like assembly J Mol Biol 377,

1544–1559

24 Tamaki N, Mizutani N, Kikugawa M, Fujimoto S &

Mizota C (1987) Purification and properties of

b-ureido-propionase from the rat liver Eur J Biochem 169, 21–26

25 Sakamoto T, Sakata SF, Matsuda K, Horikawa Y & Tamaki N (2001) Expression and properties of human liver beta-ureidopropionase J Nutr Sci Vitaminol (Tokyo) 47, 132–138

26 Matthews MM, Liao W, Kvalnes-Krick KL & Traut

TW (1992) b-Alanine synthase: purification and alloste-ric properties Arch Biochem Biophys 293, 254–263

27 Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hiroz-ane-Kishikawa T, Vandenhaute J, Orkin SH, Hill DE, van den Heuvel S et al (2004) Toward improving Caenorhabditis elegansphenome mapping with an OR-Feome-based RNAi library Genome Res 14, 2162–2168

28 Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann

M et al (2003) Systematic functional analysis of the Caenorhabditis elegansgenome using RNAi Nature

421, 231–337

29 Sonnichsen B, Koski LB, Walsh A, Marschall P, Neu-mann B, Brehm M, Alleaume AM, Artelt J, Bettencourt

P, Cassin E et al (2005) Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans Nature

434, 462–469

30 Kumar S, Aninat C, Michaux G & Morel F (2010) Anticancer drug 5-fluorouracil induces reproductive and developmental defects in Caenorhabditis elegans Reprod Toxicol 29, 415–420

31 Rawls JM Jr (2006) Analysis of pyrimidine catabolism

in Drosophila melanogaster using epistatic interactions with mutations of pyrimidine biosynthesis and b-alanine metabolism Genetics 172, 1665–1674

32 Lecroisey C, Se´galat L & Gieseler K (2007) The C ele-gansdense body: anchoring and signaling structure of the muscle J Muscle Res Cell Motil 28, 79–87

33 Vreken P, van Kuilenburg ABP, Hamajima N, Mei-nsma R, van Lenthe H, Go¨hlich-Ratmann G, Assmann

BE, Wevers RA & van Gennip AH (1999) cDNA clon-ing, genomic structure and chromosomal localization of the human BUP-1 gene encoding b-ureidopropionase Biochim Biophys Acta 1447, 251–257

34 Brenner S (1974) The genetics of Caenorhabditis elegans Genetics 77, 71–94

35 Scha¨gger H, Cramer W & von Jagow G (1994) Analy-sis of molecular masses and oligomeric states of pro-tein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis Anal Biochem 217, 220–230

36 Bolleter WT, Bushman CJ & Tidwell PW (1961) Spec-trophotometric detection of ammonia as indophenol Anal Chem 33, 592–594

37 Hiller A & van Slyke D (1933) Determination of ammo-nia in blood J Biol Chem 102, 499

38 Traut TW & Loechel S (1984) Pyrimidine catabolism: individual characterization of the three sequential enzymes with a new assay Biochemistry 23, 2533–2539

39 Naguib FNM, el Kouni MH & Cha S (1985) Enzymes

of uracil catabolism in normal and neoplastic human

tissues Cancer Res 45, 5405–5412

40 Ko¨nig S, Haegele KD & Fales HM (1998) Comment on

the cylindrical capacitor electrospray interface Anal

Chem 70, 4453–4455

41 Wolf M, Nunes F & Paul RJ (2005) Coordinates, DNA

content and heterogeneity of cell nuclei and segments of

the Caenorhabditis elegans intestine Histochem Cell Biol

124, 359–367

42 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F

& Higgins DG (1997) The ClustalX windows interface:

flexible strategies for multiple sequence alignment aided

by quality analysis tools Nucleic Acids Res 25, 4876–

4882

43 Guindon S & Gascuel O (2003) A simple, fast and

accu-rate algorithm to estimate large phylogenies by

maxi-mum likelihood Syst Biol 52, 696–704

44 Arnold K, Bordoli L, Kopp J & Schwede T (2006) The

SWISS-MODEL Workspace: a web-based environment

for protein structure homology modelling

Bio-informatics, 22, 195–201

45 Wasternack C, Lippmann G & Reinbotte H (1979)

Purification and properties of b-ureidopropionase of

Euglena gracilis Biochim Biophys Acta, 570, 341–351

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

by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors