ß-Ureidopropionase Deficiency

Biochimica et Biophysica Acta 1822 (2012) 1096–1108

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ß-Ureidopropionase deﬁciency: Phenotype, genotype and protein structural consequences in 16 patients

André B.P. van Kuilenburg a,⁎, Doreen Dobritzsch b, Judith Meijer a, Michael Krumpel b, Laila A. Selim c, Mohamed S. Rashed d, Birgit Assmann e, Rutger Meinsma a, Bernhard Lohkamp b, Tetsuya Ito f, Nico G.G.M. Abeling a, Kayoko Saito g, Kaoru Eto h, Martin Smitka i, Martin Engvall j, Chunhua Zhang k, Wang Xu l, Lida Zoetekouw a, Raoul C.M. Hennekam m a Academic Medical Center, Emma Children's Hospital, Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, 1105 AZ Amsterdam, The Netherlands b Karolinska Institutet, Dept. of Medical Biochemistry and Biophysics (MBB), S-17177 Stockholm, Sweden c Cairo University Children Hospital, Department of Pediatrics, Pediatric Neurology & Neurometabolic Divisions, Cairo, Egypt d Pharmagene Labs, Dokki 12311, Giza, Egypt e University Children's Hospital, Department of General Pediatrics, 40225 Düsseldorf, Germany f Nagoya City University, Graduate School of Medical Sciences, Department of Pediatrics and Neonatology, Nagoya 467-8601, Japan g Tokyo Women's Medical University, Institute of Medical Genetics, Tokyo 162-0054, Japan h Tokyo Women's Medical University, Department of Pediatrics, Tokyo 162-8111, Japan i University of Dresden, Children's Hospital, Department of Neuropediatrics, 01307 Dresden, Germany j Karolinska University Hospital Huddinge, Centre for Inherited Metabolic Diseases, S-141 86 Stockholm, Sweden k MILS International, Department of Research & Development, Kanazawa, 921-8154, Japan l Beijing Children's Hospital, Beijing, China m Academic Medical Center, Emma Children's Hospital, Department of Pediatrics, 1105 AZ Amsterdam, The Netherlands article info abstract

Article history: ß-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion Received 8 June 2011 of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, Received in revised form 29 March 2012 ammonia and CO2. To date, only five genetically confirmed patients with a complete ß-ureidopropionase de- Accepted 9 April 2012 ficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly Available online 14 April 2012 identified ß-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnor- Keywords: ß-Ureidopropionase mal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of UPB1 N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine and plasma. Analysis of UPB1, Neurological abnormalities encoding ß-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Het- Homology modeling erologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant Functional and structural protein analysis ß-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ß-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated. © 2012 Elsevier B.V. All rights reserved.

1. Introduction pyrimidine degradation pathway (Fig. 1) [1].Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting en- In most organisms, the pyrimidine bases uracil and thymine are zyme, catalyzing the reduction of uracil and thymine to 5,6-dihy- degraded by a three-step reaction sequence of the reductive drouracil and 5,6-dihydrothymine, respectively. The second step is catalyzed by dihydropyrimidinase (EC 3.5.2.2) and consists of a re- versible hydrolysis of the dihydropyrimidines. Finally, the resulting ⁎ Corresponding author at: Academic Medical Center, Laboratory Genetic Metabolic N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid are Diseases F0-220, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: +31 20 5665958; fax: +31 20 6962596. converted in the third step to ß-alanine and ß-aminoisobutyric acid, E-mail address: [email protected] (A.B.P. van Kuilenburg). ammonia and CO2 by ß-ureidopropionase (EC 3.5.1.6).

ß-Ureidopropionase has been purified and characterized from various eukaryotic species including rat, bovine, human and Drosophila melanogaster [15–18]. Based on comparison of primary structures and phylogenetic analysis, the eukaryotic ß-ureidopropionases can be assigned to two subfamilies [19]. One subfamily consists of mainly fungal enzymes, while higher eukaryotic ß-ureidopropionases consti- tute the second subfamily. Although functionally related, the members of the distinct subfamilies show a high degree of structural diversity [18–20]. The amino acid sequence of human ß-ureidopropionase shares 63% identity with the enzyme from D. melanogaster (DmβUP), whose crystal structure was recently determined [18]. Its subunit shows significant similarity to that of other enzymes of the nitrilase superfamily to which also human ß-ureidopropionase belongs [21]. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified patients with a complete ß-ureidopropionase deficiency and compared these to the 5 previously characterized patients. We report the functional and structural consequences of the mutations at the protein level using a homology model that was generated based on the crystal structure of recombinant DmβUP.

2. Materials and methods

2.1. Patients

The patients were collected in Germany, Sweden, Egypt, China and Japan. Criteria to test for a metabolic disorder in general or specifically for a disturbed purine/pyrimidine metabolism vary in the various countries. In general patients are tested for a metabolic disorder if patients show developmental delay, neurological manifestations (such as ataxia or dystonia), enlarged liver and spleen or gingiva hyperpla- sia, skeletal dysostosis and if patients have prolonged signs and symptoms of the hematological, immunological, ophthalmological and gastrointestinal systems. This indicates that patients with a different phenotype will not be tested and will not be detected, which creates a bias in symptomatology. Based on the above indication, 9 patients were identified in whom a ß-ureidopropionase deficiency was likely. Subsequent analysis of family members of the affected patients revealed another 2 probably affected persons. Samples of all patients were forwarded to the Laboratory for Genetic Metabolic Dis- Fig. 1. Catabolic pathway of the pyrimidine bases uracil and thymine. The genes encod- eases in Amsterdam for further analysis. ing the enzymes of the pyrimidine degradation pathway are given in parenthesis.

2.2. Analysis of pyrimidines in body fluids It is generally accepted that the pyrimidine degradation pathway is an important route for the synthesis of ß-alanine and ß-aminoisobutyric Concentrations of uracil, thymine, dihydrouracil, dihydrothymine, acid in human beings [2,3]. ß-Alanine is a structural homologue of γ- N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in aminobutyric acid and glycine, which are the major inhibitory neuro- urine, plasma and CSF were determined using reversed-phase HPLC transmitters in the central nervous system. ß-Alanine itself has been combined with electrospray tandem mass spectrometry [22,23]. suggested to be involved in synaptic transmission and has been shown to regulate dopamine levels [4,5]. ß-Aminoisobutyric acid is a 2.3. PCR amplification of coding exons of UPB1 and DNA sequencing partial agonist of the glycine receptor and stimulates the secretion of leptin and fatty acid oxidation [6–8]. Thus, an altered homeostasis of DNA was isolated from EDTA blood by standard procedures. Exons these ß-amino acids might underlie some of the clinical abnormalities 1–11 of UPB1 and their flanking sequences were amplified using the encountered in patients with a pyrimidine degradation defect [9,10]. primer sets, as described before [12]. Amplification was carried out To date, more than 75 patients have been described with a DPD in 25 μl reaction mixtures containing 20 mM Tris/HCl pH 8.4, 50 mM deficiency and 28 patients with a dihydropyrimidinase deficiency KCl, 1.5 mM MgCl2,0.4μM of each primer, 0.2 mM dNTPs and 0.02 U [10,11]. In contrast, only 5 genetically confirmed patients with a ß- of Platinum Taq polymerase. After initial denaturation for 5 min at ureidopropionase deficiency have been described until now and the 95 °C, amplification was carried out for 8 cycles (30 s 95 °C, 30 s 55 °C, clinical, biochemical and genetic spectrum in patients with a ß- 60 s 72 °C) and 27 cycles (30 s 95 °C, 90 s 72 °C) with a final extension ureidopropionase deficiency is still largely unknown [12,13].Theß- step of 10 min at 72 °C. PCR products were separated on 1.5% agarose ureidopropionase gene (UPB1) gene has been mapped to chromosome gels, visualized with ethidium bromide treated with exoSAP-IT and 22q11.2 and consists of 11 exons spanning approximately 20 kb of used for direct sequencing. genomic DNA [14]. The cDNA coding for human ß-ureidopropionase Sequence analysis of genomic fragments amplified by PCR and ex- contains an open reading frame of 1152 nucleotides, corresponding to pression plasmids was carried out on an Applied Biosystems model a protein of 384 amino acids with a calculated molecular weight of 3730 automated DNA sequencer using the Big dye-terminator method 43,158 Da [14]. (Perkin Elmer Corp., Foster City, CA, USA). UPB1 sequence of patients 1098 A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 was compared to that observed in controls and the reference sequence polymerase (Stratagene/Agilent) and the respective primers: forward: of UPB1 (Ref Seq NM_016327.2). 5′-AAG TTC TGT TTC AGG GCC CGG CGG GCG CTG AGT GGA AGT C-3′; reverse: 5′-ATG GTC TAG AAA GCT TTA CTC TTT CAC GAT GGT GGG 2.4. Expression and activity analysis of ß-ureidopropionase in GCT G-3′ (italics: vector specific5′ extensions; bold: stop codon). Escherichia coli pOPINF was digested with restriction enzymes HindIII and KpnI and purified. Subsequently, 1.0 μg digested vector and 0.5 μgpurified PCR To obtain an expression plasmid containing wild-type human ß- product were treated with 0.3 U of T4 DNA polymerase (NEB) at room ureidopropionase cDNA (pSE420-ßUP), the NcoI–NaeI insert of a temperature for 30 min. The reaction was stopped by addition of dCTP plasmid comprising the complete coding region of the human ß- (1 mM). 25 ng of T4 DNA polymerase-treated vector and 11 ng of iden- ureidopropionase gene was subcloned into the pSE420 vector [14]. tically treated insert were incubated for 30 min at 37 °C and then trans- Mutations were introduced into the human ß-ureidopropionase se- formed into E. coli strain DH5α. Positive clones were identified by blue quence using the megaprimer technique and the resulting mutant and white screening. The correct insert was confirmed by sequencing of genes were cloned into the pSE420 vector, essentially as described extracted plasmid DNA (Eurofins MWG Operon GmbH, Germany). before [12]. Resulting clones were sequenced completely to verify Point mutations were introduced by site-directed mutagenesis the presence of the mutation and to exclude the presence of random using a modified QuikChange® protocol. Briefly, plasmid DNA was am- mutations introduced by PCR artifacts. Expression plasmids were in- plified by PCR using 25 ng of wild-type plasmid DNA, 62.5 ng of the re- troduced into the E. coli strain BL21(DE3). A 25 ml LB broth culture, spective mutagenesis primer pair, 1.25 U PfuTurbo DNA polymerase, supplemented with 50 μg/ml ampicillin, was inoculated with 250 μl 200 μM dNTPs and 1× PCR reaction buffer (25 μl final volume). of an overnight preculture grown in LB broth. Cells were grown for 3 h at 37 °C and induction was performed by the addition of isopropyl-ß- Mutation Primers Primer sequences D-thiogalactoside to a final concentration of 0.4 mM. Cells were sedi- R326Q g977a 5′-cctgacagcagccagactcctgggctg-3′ mented after 3 h and washed twice with phosphate-buffered saline g977a_antisense 5′-cagcccaggagtctggctgctgtcagg-3′ T359M c1076t 5′-tgatgtctggaacttcaagatgatgggcaggtatga-3′ (PBS). The cell pellet was frozen at −80 °C and subsequently resus- c1076t_antisense 5′-tcatacctgcccatcatcttgaagttccagacatca-3′ μ pended in 300 l isolation buffer (35 mM potassium phosphate pH 7.4 L013S t38c 5′-tcgctggaggaatgctcggagaagcacctg-3′ and 2.5 mM MgCl2) and lysed by sonication. The crude lysate was cen- t38c_antisense 5′-caggtgcttctccgagcattcctccagcga-3′ trifuged at 18,000×g at 4 °C for 15 min and the resulting supernatant S264R c792a 5′-ataggagcactcagagagtccctgtggcc-3′ ′ ′ was stored at −80 °C. c792a_antisense 5 -ggccacagggactctctgagtgctcctat-3 G235R g703a 5′-gtgaacatttgctacaggcggcaccaccc-3′ The activity of ß-ureidopropionase was determined at 37 °C in a g703a_antisense 5′-gggtggtgccgcctgtagcaaatgttcac-3′ standard assay mixture containing an aliquot (8–10 μl) of cell superna- R236W c706t 5′-catttgctacgggtggcaccaccccct-3′ tant, 200 mM MOPS (pH 7.4), 1 mM dithiothreitol and 500 μM[14C]-N- c706t_antisense 5′-agggggtggtgccacccgtagcaaatg-3′ carbamyl-ß-alanine [24]. Separation of radiolabeled N-carbamyl- ß-alanine from radiolabeled ß-alanine was performed isocratically Following PCR with modified cycling parameters (95 °C 1 min; fl (50 mM NaH2PO4,pH4.5,ata ow rate of 1 ml/min) by reversed- 15 cycles: 95 °C 1 min, 55 °C 1.5 min, 68 °C 6 min 44 s; 68 °C 20 min), μ phase HPLC on an Aqua C18 column (250×4.6 mm, 5 m particle size, 5 U DpnI was added to each reaction and incubated at 37 °C for 1 h. Phenomenex, Torrance, CA) and a guard column (Securityguard C18, The DNA was transformed into E. coli DH5α. The identity of the single 4×3.0 mm ID, Phenomenex, Torrance, CA) with on-line detection of base substitutions was confirmed by sequencing. the radioactivity.

2.5. Western blot analysis 2.7. Protein expression and puriﬁcation

Supernatants (10 μg) were fractionated on a 8% (w/v) SDS- Plasmids containing wild-type or mutant UPB1 were transformed polyacrylamide gel and transferred to a nitrocellulose ﬁlter. Blocking into E. coli strain C41(DE3)pRARE2. 1.5 l of culture broth (2.4% (w/ of the membrane was performed for 16 h at 4 °C with PBS (pH 7.4) v) yeast extract, 1.2% (w/v) peptone, 0.1 M potassium phosphate containing 5% (w/v) milk powder. Subsequently, the membrane was (pH ~7.2), 10 mM NaCl, 10 mM NH4Cl, 2 mM MgSO4) was inoculated incubated for 1 h with a 1:1000 dilution of anti-UPB1 antibody with 15 ml overnight culture in LB medium. All media were supple- (Sigma Aldrich B.V., Zwijndrecht, the Netherlands) in PBS with 5% mented with 100 μg/ml carbenicillin and 35 μg/ml chloramphenicol.

(w/v) milk powder, supplemented with 0.05% (v/v) Tween 20. The Cells were grown under agitation at 37 °C until OD600 ~0.6 was membranes were washed three times (10 min each), once with PBS reached, cooled for ~30 min at 4 °C, and induced with IPTG (0.5 mM containing 0.05% (v/v) Tween 20, 5% (w/v) milk powder and twice final concentration). After further incubation under agitation for with PBS containing 0.05% (v/v) Tween 20 and incubated for 1 h 20 h at 25 °C, cells were harvested by centrifugation at 4000×g at with PBS containing 0.05% (v/v) Tween 20, 5% (w/v) milk powder 4 °C. The cells were washed with cold buffer containing 50 mM Na- and a 1:2000 dilution of a polyclonal goat anti-rabbit secondary anti- HEPES pH 7.4, 100 mM NaCl. After centrifugation the pellet was resus- body conjugated to alkaline phosphatase (Santa Cruz Biotechnology, pended in cold cell lysis buffer (50 mM Na-HEPES pH 7.4, 300 mM Inc, Santa Cruz, CA). After rinsing the membrane three times (10 min NaCl) supplemented with 50 μg/ml lysozyme, 10 μg/ml DNaseI and one each), twice with PBS containing 0.05% (v/v) Tween 20 and once with ROCHE “Complete®” EDTA-free protease inhibitor cocktail tablet, to a PBS, detection was performed with BCIP/NBT (Promega Benelux B.V., final volume of ~35 ml. Cells were disrupted by incubation at 25 °C for Leiden, The Netherlands). 20 min, followed by one freeze/thaw cycle and sonication. The supernatant resulting from centrifugation (40 min, 12,000×g, 4 °C) was stored at 2.6. Molecular cloning and site-directed mutagenesis −80 °C until further use. The supernatant was supplemented with 25 mM imidazole and The coding sequence of the human UPB1 gene was re-cloned into 500 μl Ni-NTA agarose affinity matrix slurry (50% suspension, Qiagen) the pOPINF [25] expression vector (Oxford Protein Production Facility, per 50 ml lysate. After incubation for 1 h at 4 °C under rotation, the ly- GenBank Accession No. EF372398), which includes the coding sequence sate mixture was sedimented in a gravity flow column and washed for a cleavable N-terminal 6×His-fusion tag. To this end, a sequence in- with cell lysis buffer containing increasing imidazole concentrations dependent cloning approach (SLIC) was utilized with pSE420-ßUP as a (25–75 mM). β-Ureidopropionase was eluted from the column using template. The DNA was amplified using high-fidelity PfuTurbo DNA a stepwise gradient of 100–500 mM imidazole in cell lysis buffer. A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 1099

β-Ureidopropionase-containing fractions were collected and con- the presence of 10 mM DTT for ~16 h at 4 °C. To remove uncleaved pro- centrated to 10 ml final volume (Vivaspin 20, 10 kDa MW cutoff, tein, the 3C protease and impurities, 500 μl Ni-NTA matrix slurry was PES, Sartorius Stedim Biotech, Germany). The buffer was exchanged to added to the solution and sedimented in a gravity flow column. The buffer A (20 mM Na-HEPES pH 7.4, 50 mM NaCl) using a Sephadex- flow-through was concentrated to 5 ml final volume and applied to a G25 desalting column (HiPrep™ 26/10, Pharmacia Biotech). Protein 1 ml HiTrap Q HP column equilibrated with buffer A. Wild-type and mu- containing fractions were collected and diluted to 50 ml with buffer A. tant β-ureidopropionase were eluted with a linear gradient from 0 to Cleavage of the N-terminal His-tag was achieved by addition of 1 μg 100% buffer B (20 mM Na-HEPES pH 7.4, 800 mM NaCl) over 18 column His-tagged 3C protease per 1 mg of target protein and incubation in volumes with a flow rate of 1 ml/min. β-Ureidopropionase-containing

Fig. 2. Sequence conservation of the mutation sites in higher eukaryotic β-ureidopropionases. The alignment of the amino acid sequences of human (Hs), orangutan (Pp), mouse (Mm), rat (Rn), Drosophila melanogaster (Dm) and Arabidopsis thaliana (At) β-ureidopropionase reveals the strict conservation of all but one mutation sites. Bold font is used for the mutation sites. Sequence conservation at these sites is indicated by gray background coloring. An asterisk (*) indicates fully conserved positions. Colon (:) and period (.) indicate positions at which strongly or weakly similar properties are conserved, respectively. 1100 A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 fractions were combined and concentrated to a final concentration of β-ureidopropionase from D. melanogaster (PDB ID: 2vhi and 2vhh) ~10 mg/ml, aliquoted, flash frozen and stored at −80 °C until further [18]. The single amino acid exchanges caused by the point mutations use. were manually introduced to the homology model using WinCoot [26], choosing energetically preferred side chain conformations that 2.8. Dynamic light scattering and circular dichroism spectroscopy cause the least steric clashes and interact optimally with surrounding residues. A model of a (putative) tetramer of human β-ureidopropionase Dynamic light scattering (DLS) measurements were conducted was constructed from monomeric homology models by superposition using a PD2000DLS plus Precision Dynamic Light Scattering detector onto the respective subunits of DmβUP in WinCoot. The figures showing (Precision Detectors, Bellingham, MA, USA). The sample volume was the point mutation sites and their environment were generated using the 20 μl containing 2 mg/ml enzyme variant in 20 mM Na-HEPES buffer program PyMOL (DeLano, W.L. The PyMOL Molecular Graphics System pH 7.4, 100 mM NaCl. Measurements were carried out 150 times at (2002), url: http://www.pymol.org). The sequences shown in Fig. 2 20 °C. Prior to measurement all samples were centrifuged for 15 min at were aligned with CLUSTAL W [27]. 16,000×g and 4 °C and filtered through a 0.1 μm filter to remove large aggregates. For wild-type β-ureidopropionase, the equivalent measure- 3. Results ments were also performed at a protein concentration of 15 mg/ml. Circular dichroism (CD) measurements were conducted on a 3.1. Clinical phenotype JASCO J-810 spectropolarimeter using a cuvette of 0.1 cm path length. The samples contained 0.2 mg/ml enzyme variant in 2 mM Na-HEPES pH The clinical features of all 16 known ß-ureidopropionase deficient 7.4, 10 mM NaCl. The spectra were recorded in the range of 190–260 nm patients are shown in Table 1. The sex ratio was equal. Most patients at 20 °C and corrected for background (buffer). Ten scans were averaged were referred in the first two years of life; in two families an adult without smoothing. was only recognized after diagnosis in a relative. The most common findings were an abnormal MRI, seizures, intellectual disabilities 2.9. Native gel electrophoresis and hypotonia or hypertonia (in 85%, 60%, 57% and 53% of the patients, respectively). MRI findings varied from agenesis of the callosal Native polyacrylamide gel electrophoresis was performed using body and vermis hypoplasia with brain stem hypoplasia to delayed 4–20% Mini-Protean® TGX™ Precast Gels (BIO-RAD). The electrode myelination and general brain atrophy. Two siblings had cortical dys- (running) buffer was 25 mM Tris, 192 mM glycine pH 8.3. The sample plasia. In four patients convulsions occurred as infantile spasms, buffer included 62.5 mM Tris pH 6.8, 40% glycerol, and 0.01% bromo- which changed into focal and tonic–clonic seizures in one of them. phenol blue. Gels were run at 25 °C, and the voltage was kept con- Three other patients also had focal epilepsy. Growth retardation, mi- stant at 200 V. crocephaly and autism were observed less frequently. Three patients developed opticus atrophy with a decreased vision. One other patient 2.10. Homology-based structural analysis had bilateral microphthalmia and, likely, secondary optical atrophy. Two patients showed a progression in symptomatology, especially A homology model of human β-ureidopropionase was generated with respect to their neurological symptoms. Phenotypic variability using SWISSMODEL routines and the available crystal structure of of ß-ureidopropionase deficiency was demonstrated in one family in

Table 1 Phenotype of 16 patients from 13 families with ß-ureidopropionase deﬁciencya.

Patient Origin Consanguinity Age Gender Growth Microcephaly Intellectual Abnormal MRIb Seizuresc Hypotonia Autism Otherd (years) retardation disability

1. Japan − 0.5 F −−+ − I − +1 2.1. Turkey + 0.8 M −−− unk F + − 2.2. 30 F −−− unk −− − 3.1. Egypt + Birth F − + − CD I −−3.2 3.2. Birth M − + unk CD I, F, TC −e unk 3.3. 27 M unk unk unk unk unk unk unk 4. Egypt + 0.8 M −−+ A + ++ unk 4 5. Pakistan + 2.0 F + ++ ++ A, CH − ++ + 5 6. China − 1.1 M + + ++ DM −− −6 7. Germany − 0.9 F −−− AF++− 7 8. China 3.0 M + − + −−−− 9. Turkey + 5.3 F −−++ A, VA, BH, DM, − ++ unk 9 CH 10. Turkey + 3.0 F + − +DMI−−10 11. Germany − 0.9 M − + ++ A, SH F, TC ++ unk 11 12. ‘African’ − 1.0 F −−−−SAS −e unk 12 13. Australia − 1.0 M −−−−−−unk 13 Total 26% (4/15) 33% (5/15) 57% (8/14) 85% (11/13) 60% 53% 22% (9/15) (8/15) (2/9)

a − absent; + present, mild; ++ present, severe; unk unknown. b CD, cortical dysplasia; A, atrophia cerebri; DM, delayed myelination; VH, vermis hypoplasia; BH, brainstem hypoplasia; CH, callosal body hypoplasia; SH, subdural hematoma. c F, focal seizures; I, infantile spasms; TC, tonic–clonic seizures; SAS, single atonic seizure (30 min). d1Progressive mood changes [32]; 3.2facial morphology: prominent metopic suture, dolicocephaly, downward slanted palpebral fissures; recurrent episodes of desaturation unrelated to seizure activity; demise at 18 days; 4recurrent attacks of vomiting and severe dehydration, respiratory infections, demise at 13 months; 5bilateral microphthalmia, marked hyperopia, pale optic nerves; normal ERG and VEP; 6nephrotic syndrome at 1.9 years; 7initial development (until 11 months) normal; transient cerebral blindness. At 11 months bout of fever and diarrhea, development of uncontrollable seizures and cerebral edema, followed by brain atrophy; 9from birth on dystonia, abnormal eye movements including nystagmus, tremor; optic atrophy and at 2.3 year abnormal retina pigmentation, from 11 m on scoliosis, no speech [34,47]; 10possibly hearing deficit; severe speech delay [12]; 11at 5 m polytopic myoclonic jerks; no visual contact, mild optic atrophy [12,48]; 12brother died at 9 m after number of convulsive crises (no further information) [12]; 13bladder extrophy, bifidphallus,bifid scrotum, duplicated distal colon, anal atresia [13]. e Hypertonia. A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 1101 which the index patient was clinically affected whereas the same ge- concentration observed in urine of controls. In plasma, the mean con- notype did not lead to overt symptoms in the mother of the patient. centrations of N-carbamyl-ß-alanine (17±6 μM) and N-carbamyl-ß- aminoisobutyric acid (24±10 μM) concentrations were 91-fold and 178-fold, respectively, higher compared to the mean concentration ob- 3.2. Biochemical phenotype served in controls. In ß-ureidopropionase deficient patients, a strong correlation existed between urinary concentration of N-carbamyl-ß- As part of a selective screening program for inborn errors, the py- alanine and N-carbamyl-ß-aminoisobutyric acid (R2=0.80, P=0.0004). rimidine bases, uracil and thymine, and their degradation products A similar correlation existed between the N-carbamyl-ß-alanine and N- were analyzed in urine, plasma and CSF samples. Urine and plasma carbamyl-ß-aminoisobutyric acid concentration in plasma (R2 = samples of the patients showed strongly elevated levels of N-carbamyl- 0.82, P=0.001). Thus, the observed N-carbamyl-ß-amino aciduria in ß-alanine and N-carbamyl-ß-aminoisobutyric acid and moderately ele- these patients strongly suggested ß-ureidopropionase deficiency. vated levels of dihydrouracil and dihydrothymine (Fig. 3). The mean concentration of uracil (22.4±16.1 μmol/mmol creatinine), thymine (3.0± 2.1 μM μmol/mmol creatinine), dihydrouracil (58±36 μmol/mmol cre- 3.3. Genotype atinine) and dihydrothymine (134±67 μmol/mmol creatinine) was 2-fold, 6-fold, 9-fold and 43-fold, respectively, higher compared to the Analysis of the genomic sequences of exons 1–11 of UPB1 includ- mean concentration in urine of controls. In contrast, the mean concentra- ing their flanking intronic sequences in the hitherto uncharacterized tions of N-carbamyl-ß-alanine (755±361 μmol/mmol creatinine) and patients showed two previously described splice-site mutations (c.105- N-carbamyl-ß-aminoisobutyric acid (533±214 μmol/mmol creatinine) 2A>G and c.917-1G>A) [12] and 7 novel mutations, including 6 missense were 69-fold and 269-fold, respectively, higher compared to the mean mutations and one splice-site mutation (Table 2). Homozygosity for one

Fig. 3. Concentrations of N-carbamyl-ß-amino acids and dihydropyrimidines in ß-ureidopropionase deﬁcient patients and controls. Panel a shows the urinary levels of N-carbamyl- ß-alanine (NCß-Ala) and N-carbamyl-ß-aminoisobutyric acid (NCß-AIB) and panel b shows the concentrations of dihydrouracil (DHU) and dihydrothymine (DHT) in urine. The plasma levels of the N-carbamyl-ß-amino acids and dihydropyrimidines are shown in panels c and d, respectively. The top, bottom and line through the middle of a box correspond to the 75th percentile, 25th percentile and 50th percentile, respectively. The whiskers on the bottom extend from the 2.5th percentile and top 97.5th percentile. 1102 A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108

Table 2 Results of mutation analysis in 11 patients with ß-ureidopropionase deﬁciency.

Patient Genotype Effect Location

1. c.[977G>A]+[977G>A] p.[R326Q]+[R326Q] Ex 9 2.1 c.[1076C>T]+[1076C>T] p.[T359M]+[T359M] Ex 10 2.2 c.[1076C>T]+[1076C>T] p.[T359M]+[T359M] Ex 10 3.1 c.[105-2A>G]+[105-2A>G] Splicing Intron 1 3.2a 3.3 c.[105-2A>G]+[105-2A>G] Splicing Intron 1 4.lb c.[38T>C]+[38T>C] p.[L13S]+[L13S] Ex 1 5. c.[792C>A]+[873+1G>A] p.[S264R]+splicing Ex 7, intron 7 6. c.[977G>A]+[977G>A] p.[R326Q]+[R326Q] Ex 9 7. c.[703G>A]+[917-1G>A] p.[G235R]+splicing Ex 6, intron 8 8. c.[706C>T]+[792C>A] p.[R236W]+[S264R] Ex 6, Ex 7

a No DNA available. b No DNA available, parents of the deceased patient were heterozygous for the c.[38T>C] mutation.

fi Fig. 5. ß-Ureidopropionase activity and immunoblot analysis of wild-type and mutant of the identi ed mutations was observed in 7 out of 10 patients. The mu- ß-ureidopropionases expressed in E. coli as well as that of human liver. The results rep- tations c.792C>A and c.977G>A were each present in two unrelated pa- resent the mean of three independent measurements, the error bar represents 1 SD. tients. Fig. 4 shows the distribution of UPB1 mutations over the exons. Mutations were found to occur in all exons except exons 3–5. the soluble proteins. p.R236W could be purified to only ~80% homogene- 3.4. Functional analysis of mutant ß-ureidopropionase enzymes ity, due to lower expression levels, all other enzyme variants to >95% homogeneity. The β-ureidopropionase activity of the wild-type and pu- To investigate the effect of the 6 missense mutations on the activ- rified mutant proteins is given in Table 3. ity of ß-ureidopropionase, the mutations were introduced into a CD spectra were recorded to investigate whether the reduced en- pSE420-ßUP vector using the megaprimer technique and expressed in zymatic activity of the mutant enzymes was caused by folding defects. E. coli. No endogenous ß-ureidopropionase activity (b0.3 nmol/mg/h) The CD spectra of p.L13S and p.S264R were nearly identical to that of could be detected in the E. coli strain used for the expression of the con- wild-type β-ureidopropionase in the far UV range, indicating no or lit- structs. Introduction of wild-type ß-ureidopropionase construct in- tle disturbance of the secondary structure by the single amino acid ex- creased ß-ureidopropionase activity more than 1000-fold above the changes (Fig. 1S, Supplementary data). In contrast, while still clearly background level. Expression of ß-ureidopropionase constructs con- indicating that the proteins were folded, the spectra of p.R236W and taining the mutations p.L13S, p.G235R, p.R236W, p.S264R, p.R326Q in particular p.T359M showed larger deviations from the wild-type and p.T359M yielded no or significantly decreased ß-ureidopropionase β-ureidopropionase spectrum. activity (Fig. 5). Size exclusion chromatography experiments originally included in To exclude the possibility that the lack of ß-ureidopropionase activ- the purification procedure for wild-type β-ureidopropionase did not ity was the result of an inability to produce the mutant protein in E. coli, indicate the presence of large amounts of protein aggregates or of the expression levels were analyzed by immunoblotting. Fig. 5 shows an oligomer equilibrium. These experiments were usually performed that the mutant proteins were expressed in comparable amounts as at lower protein concentrations (b1 mg/ml), and the retention time the wild-type protein. Thus, the lack of ß-ureidopropionase activity of of the single protein peak with β-ureidopropionase activity would mutant ß-ureidopropionase enzymes in E. coli is not due to rapid degra- correspond to a monomeric or dimeric state of the enzyme. dation of the various mutant ß-ureidopropionase proteins in the E. coli In contrast, the dynamic light scattering experiments performed supernatants. Nevertheless, it does not give sufficient evidence as to at a protein concentration of 2 mg/ml revealed that wild-type as whether the mutant proteins are stably folded. well as mutant β-ureidopropionase solutions showed a high degree To enable rapid purification and thus further analysis of the mutant of polydispersity likely to be caused by the concomitant presence of enzymes, the human UPB1 gene was re-cloned into a vector (pOPINF) different oligomeric states (Fig. 2S, Supplementary data). In addition, fusing a His-tag at the N-terminus of the protein. The subsequent intro- the observation of very large hydrodynamic radii indicated that all of duction of single point mutations using the QuikChange® method was the purified β-ureidopropionase proteins have a tendency to aggre- straightforward for all but one of the sites. Several attempts to mutate gate — although visible precipitation was absent. Both observed phe- the site giving rise to p.R326Q each time resulted in amplification of in- nomena together led to a large variation between the five repeated complete vectors for yet unknown reasons. No sufficient expression of measurements per protein variant and prevented an accurate estimation soluble protein was observed for p.G325R preventing further purifica- of the predominant oligomeric state. Nevertheless, while for wild-type tion. The amounts of purified protein obtained from the same culture β-ureidopropionase hydrodynamic radii of 4.0–5.5 nm (average 4.9 nm) volume ranked according to p.R236W b p.S264R b p.L13S b wild-type b were most often observed, possibly corresponding to homodimeric to p.T359M, giving a crude estimate of therelativeexpressionlevelsfor homohexameric assemblies, smaller average radii were measured for

Fig. 4. Genomic organization and mutation analysis of the UPB1 gene. UPB1 consists of 11 exons encoding an open reading frame of 1152 bp (depicted in gray). The various mutations identiﬁed in ß-ureidopropionase deﬁcient patients are indicated, numbers correspond to the cDNA position. A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 1103

Table 3 difficult to predict if their assembly does not engage the same subunit Activity of purified wild-type and mutant ß-ureidopropionases. surfaces as seen for DmβUP. Enzyme ß-Ureidopropionase activity (nmol/mg/h) Interestingly, all six point mutations affect residues that are well (R326) or strictly (L13, G235, R236, S264, T359) conserved in higher Wild-type 16,919±638 p.R236W n.d. (b9.5 nmol/mg/h) eukaryotic ß-ureidopropionases (Fig. 2) and cluster at or close to the p.T359M n.d. (b4.9 nmol/mg/h) subunit surface that becomes buried upon dimerization (Fig. 6b). Only p.L13S 1003±110 three mutations, G235R, R236W and S264R, lead to amino acid ex- p.S264R 3346±51 changes in the active site and are, therefore, likely to have also direct ef- n.d., not detectable. fects on substrate binding and catalysis. G235 is close to the catalytically crucial cysteine (C233) that per- forms the nucleophilic attack on the substrate resulting in the cova- the mutants p.L13S (2.8 nm), p.S264R (3.4 nm) and p.T359M (2.8 nm) lent acyl-enzyme complex [18]. The mutation G235R introduces a at the same protein concentration. It indicates a shift of a potential equi- large amino acid side chain for which there is no space available at librium of oligomeric states towards the lower molecular weight this location. The larger structural rearrangements in the active site species, which is confirmed by native gel electrophoresis (Fig. 3S, Sup- cavity required to prevent clashes with surrounding residues are plementary data). p.L13S and p.T359M appear as a single broad band expected to lead to enzyme inactivity and misfolding and defects in of lower molecular weight than the sharper double band observed for oligomerization (Fig. 7a). The inability to obtain significant expres- the wild-type enzyme. Native gel analysis of p.S264R reveals two distinct sion of soluble protein for this mutant supports this conclusion. double bands, one with approximately the same and one of lower molec- R236 is part of the active site and has a putative role in binding the ular weight than that observed for the wild-type β-ureidopropionase. In carboxyl group of the substrate (Fig. 7b). The side chain of the trypto- contrast, a larger average hydrodynamic radius (8.3 nm) was observed phan introduced by the mutation R236W is much more rigid and hy- for p.R236W. This may be attributed to the presence of a contaminating drophobic and does not carry a charge at physiological pH. Therefore, protein since analysis by native gel electrophoresis does not give any in- the shape and charge state of the active site would be significantly al- dication for a stabilization of higher oligomeric states or increased aggre- tered by the mutation, preventing substrate binding and rendering gation. Instead, the predominant state of this mutant is smaller in size the enzyme inactive. Furthermore, a multitude of close contacts to than that of the wild-type enzyme. DLS measurements performed for nearby residues are likely to cause protein misfolding. The clear but wild-type β-ureidopropionase at a protein concentration of 15 mg/ml relatively small changes in the CD spectrum of p.R236W indicate led to a slight increase in observed hydrodynamic radius (5.3 nm) and that misfolding is locally restricted, most likely to the direct neighbor- polydispersity. hood of the mutation site. Nevertheless, such smaller structural deviations could potentially impair the formation of higher oligomers due 3.5. Structural effects of ß-ureidopropionase mutations to the proximity of the mutation site to subunit surfaces that are in DmβAS involved in dimerization and tetramerization. To further investigate the effects of the missense mutations iden- The mutation S264R abolishes the hydrogen bond to Y314, which tified in the human UPB1 gene, a homology model of the gene product may be important for structural fixation of a residue stretch that is in- was generated using the 3D structure of ß-ureidopropionase from volved in shaping the entrance to the active site (Fig. 7c). Most steric D. melanogaster (DmβUP) (Fig. 6a) [18]. The 63% sequence identity clashes with adjacent residues can be avoided if the introduced argi- shared by both enzymes indicates that the αββα-sandwich core of the nine side chain is oriented towards V202, F205, I260, W354 (from a subunit is structurally well conserved while peripheral structure ele- neighboring subunit) and T259. However, this would obstruct the ments could show deviations. It is not yet known whether the physio- tunnel leading from the protein surface to the active site and thereby logically relevant oligomeric assembly of human ß-ureidopropionase hinder substrate binding, and also be energetically unfavorable due to mirrors that of DmβUP. Hence, the consequences of mutations affecting the placement of a hydrophilic and charged group in a hydrophobic surfaces buried upon formation of oligomers larger than a dimer are environment. Based on the homology modeling, mutation-induced

Fig. 6. The crystal structure of DmβUP and location of the mutation sites. (a) Schematic view of the homooctameric DmβUP in cartoon representation with each subunit colored differently. (b) Schematic view of a dimeric unit. For one of the subunits β-strands are depicted in brown and helices in salmon; the other subunit in the dimer is colored cyan. The mutation sites identified for human β-ureidopropionase are indicated by space-fill models of the corresponding amino acid side chain, shown in red for one subunit and in blue for the other. The labels state first the site in the human enzyme, then the corresponding DmβUP site. The location of the active site is indicated by a space-fill model of the active site cysteines (C233 in human β-ureidopropionase, C234 in DmβUP) colored yellow for both subunits. 1104 A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108

Fig. 7. The environment of the mutation sites G235R, R236W and S264R in the homology model of human β-ureidopropionase. The dimeric model of the human β- ureidopropionase is shown in cartoon representation with different colors for distinct subunits. Stick models of the side chains introduced by the point mutations are shown with carbon atoms in magenta. The conformer causing the least clashes with surrounding residues was chosen. The replaced native side chains are shown with yellow carbon atoms and thinner sticks. The catalytic cysteine is shown in ball-and-stick with yellow carbon atoms. Further residues discussed in the text or within van-der-Waals radius of the mutation site are shown with carbon atoms in white for the subunit depicted in salmon, and in light blue for the (blue) partner subunit in the dimer (labeled with an asterisk). Amino acid side chains of DmβUP that are either not conserved in human β-ureidopropionase, differently oriented or involved in different interactions are shown as stick models in green. In these cases, labels name ﬁrst the residue present in human β-ureidopropionase, followed by the DmβUP residue in brackets. Hydrogen bonds are indicated by dotted black lines, and surfaces outlining the dimensions of the active site cavity and the entrance area are shown in light gray. (a) Close-up stereo view of the G235R mutation site. (b) Close-up stereo view of the R236W mutation site. C233, K196 and E119 form the catalytic triad present in all nitrilase-like enzymes and interact with the carbamoyl moiety of the substrate. This triad is in most but not all branches of the nitrilase superfamily extended to a tetrad by a conserved glutamate [27] corresponding to E207 in human β-ureidopropionase. R236, T259 and H237 are thought to be important for the recognition and binding of carboxyl group of the substrate [18]. (c) Close-up stereo view of the S264R mutation site.

structural changes would thus be expected to be of larger scale and prob- collapse of the 4-helical bundle and significantly destabilize the mono- ably also affect dimer or higher oligomer assembly, since S264 is located mer–monomer interface. However, the CD spectrum of p.L13S is basi- at the interface between monomers. Although differences in the far-UV cally unaltered, arguing against extensive changes of the secondary CD spectra between p.S264R and wild-type β-ureidopropionase are min- and tertiary structures of the mutant protein. The spatial distance be- imal, the decrease in hydrodynamic radius for p.S264R and the appear- tween mutation and active site also excludes direct interference of the ance of an additional band for a lower molecular weight species in the amino acid exchange with substrate binding or the catalytic residues. native gel would support the latter conclusion. Another explanation for the significant decrease in enzymatic activity L13 belongs to the ~65 amino acid extension at the N-terminus of p.L13S is its potential dependence on proper oligomer assembly, that is unique to the ß-ureidopropionases branch of the nitrilase whichisaffectedbythemutation. superfamily. It prohibits higher oligomer assembly modes that are The effects of the R326Q exchange are difficult to predict. In DmβUP, typical for members of other branches but distinctly different from the corresponding R327 is located near the dimer interface without that of DmβUP. The L13 side chain contributes to the densely packed being directly involved in contacts with the partner subunit (Fig. 8b). hydrophobic interaction surface between the subunits of a dimer Its side chain forms hydrogen bonds to T328 and S330, of which the lat- (Fig. 8a). The non-conservative mutation to the smaller serine leads to ter is not conserved in human ß-ureidopropionase due to the replace- significant gaps in the hydrophobic core and introduces a polar side ment of S330 by a glycine. The mutation R326Q is likely to bring the chain in a non-polar environment, which may cause a hydrophobic introduced glutamine carboxamide group in close contacts with either A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 1105

Fig. 8. The environment of the mutation sites L13S, R326Q and T359M in the homology model of human β-ureidopropionase. Depicted residues are colored, represented and labeled as in Fig. 2. (a) Close-up stereo view of the L13S mutation site. Van-der-Waals surfaces of residues near the mutation site are depicted in light gray and blue to visualize the tight packing of the hydrophobic side chains. In one subunit the van-der-Waals surface of L13 is shown (yellow), in the other the corresponding surface of S13 is introduced by the mutation (magenta). (b) Close-up stereo view of the R326Q mutation site. (c) Close-up stereo view of the T359M mutation site. M212 belonging to another dimeric unit (shown in orange) is labeled with a hash. Sequence differences between human ß-ureidopropionase and DmβUP near the mutation site are here indicated by green spheres for conservative exchanges and red spheres for non-conservative exchanges. (d) Stereo view of the location of the T359M in a tetrameric homology model of human β-ureidopropionase. One subunit is shown in cartoon representation, with β-strands in brown and helices in salmon. The mutation site is indicated by the space-ﬁll model of the corresponding side chain in red, the location of the active site by a space-ﬁll model of C233 in yellow. The partner subunit in the dimer and both subunits of the other dimer are shown in surface representation in cyan and light gray, respectively.

R327 or Y30. It would furthermore eliminate the hydrogen bond to Fig. 8c and d depict the structural context of the mutation site T327 as well as stacking interactions with Y318. Thus, a glutamine T359 in the homology model of a (putative) tetramer of human ß- may only partly or not at all fulfill the structural role of R326 and there- ureidopropionase, which was constructed assuming that its assembly by affect proper dimer assembly. Purification and further characteriza- mode is equivalent to that of DmβUP. T359 precedes the C-terminal tion of the mutant enzyme are required for a precise determination of helix that interacts with its counterpart from the dimer mate in an the functional and structural consequences of the amino acid exchange. interlocking manner. It is in direct contact to residues from the partner 1106 A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 subunit by forming hydrogen bonds to H198 and H238. An additional not only a partial agonist of the glycine receptor [8] but it also increases hydrogen bond may be formed with N355. T359 is also spatially close the secretion of leptin and stimulates fatty acid oxidation [6,7].Leptinit- to residues from another dimer unit, in particular M212. The T359M self has been shown to have neuroprotective, cognitive and anticonvul- mutation introduces a larger and hydrophobic side chain, abolishing sant effects [35–37]. Thus, it is conceivable that the altered homeostasis the hydrogen bond to N355 and causing close contacts with E213, of ß-aminoisobutyric acid in patients with ß-ureidopropionase deficien- I199 and R201 from the dimer mate and with M212 from another cy might affect leptin levels and fatty acid oxidation and contribute to the dimer. Thus, also the T359M point mutation is likely to lead to a desta- development of intellectual disability and hypotonia in the patients. Nev- bilization of subunit interfaces within the dimer or between dimeric ertheless, the fact that the clinical presentation of patients with a ß-urei- units in potentially existing larger oligomers, which is supported by dopropionase deficiency seems to be different compared to that observed the results of DLS measurements and native gel electrophoresis. Devia- for patients with a DPD and dihydropyrimidinase deficiency, suggests tions from wild-type β-ureidopropionase in the CD spectrum indicate that additional factors are likely to be involved. structural changes that may range from a simple detachment of the C- In this respect, it has been suggested that N-carbamyl-ß-alanine, terminal helix and tail to dissociation of dimeric units. The complete one of the accumulating substrates, might function as an endogenous loss of enzymatic activity upon mutation of a solvent-exposed residue neurotoxin [38] as high concentrations of N-carbamyl-ß-alanine distant from the active site would rather speak for the latter. inhibited the mitochondrial energy metabolism which was accompanied by the induction of oxidative stress. Previously, however, we 4. Discussion showed that the concentrations of N-carbamyl-ß-alanine were only marginally elevated in CSF of patients, arguing against a direct role ß-Ureidopropionase deficiency is an inborn error of the pyrimidine of N-carbamyl-ß-alanine in the neuropathology [12]. Furthermore, degradation pathway, affecting the cleavage of N-carbamyl-ß-alanine two patients (2.2 and 3.2) who presented with low urinary levels of and N-carbamyl-ß-aminoisobutyric acid. Patients with complete ß- N-carbamyl-ß-amino acids were asymptomatic and severely affected, ureidopropionase deficiency presented with strongly elevated levels respectively. of the N-carbamyl-ß-amino acids in urine and plasma. It has been Mutation analysis of UPB1 showed 7 novel mutations, including 6 shown that N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric missense mutations and one splice-site mutation. The c.873+1G>A acid are weak inhibitors of dihydropyrimidinase [28] which might ex- mutation is located in a conserved splice-donor site and most likely plain the moderately increased levels of dihydrouracil and dihydrothy- leads to alternative splicing of the pre-mRNA and thus to a non- mine in the patients. However, the mean urinary concentration of functional protein. The two previously indentified splice-site muta- dihydrouracil and dihydrothymine and the pyrimidine bases uracil tions c.105-2A>G and 917-1G>A led to a variety of alternative and thymine are 5.5-fold, 1.5-fold, 2.7-fold and 15-fold, respectively, splice-variants, with deletions of a single or several exons [39]. Heter- lower in patients with a ß-ureidopropionase deficiency compared to ologous expression of 6 mutant enzymes in E. coli showed that all that observed in patients with a dihydropyrimidinase deficiency [10]. missense mutations yielded mutant ß-ureidopropionase proteins The most frequently observed clinical manifestations in the present with no or significantly decreased activity. study group were mainly neurological: MRI abnormalities, seizures, in- Recently, the crystal structure of D. melanogaster (DmβUP) has tellectual disabilities and abnormal tonus regulation. Although neuro- been determined [18]. Its subunit shows significant similarity to that logical abnormalities are also observed in patients with a DPD or of other enzymes of the nitrilase superfamily [21] and comprises a dihydropyrimidinase deficiency [9–11,29], the clinical presentation of central β-sandwich flanked on both sides by several α-helices. the patients with a ß-ureidopropionase deficiency seems to be more However, compared to other nitrilase-like enzymes the sequence of severe. The high frequency of gastrointestinal problems, which was ap- ß-ureidopropionases is extended at the N-terminus by ~65 amino parent in dihydropyrimidinase deficient patients, was not observed in acids, which in DmβUP form three α-helices and the first strand of patients with ß-ureidopropionase deficiency. The frequent occurrence one of the β-sheets. Upon dimerization, the first two helices interact of optic nerve hyoplasia going along with visual disturbances is not with their counterparts to form a 4-helical bundle located on a mirrored in the eye findings in other pyrimidine disturbances [9,10]. solvent-exposed face of the dimer. Besides this extra contribution to Malformations elsewhere were uncommon. It should be mentioned the monomer–monomer interface, the dimer is always assembled in that the present phenotype may represent a bias, due to the selection an identical fashion in nitrilase-like enzymes of known structure, oc- of patients of whom samples were submitted. The unexpected finding cluding the same face of the β-sandwich. It is therefore likely that of a pyrimidine metabolism disturbance in patients with Miller syn- human β-ureidopropionase uses a very similar assembly mode. Analy- drome characterized by missing fingers and toes, and markedly abnor- sis of the three-dimensional structure showed that the mutations mal face but no neurological problems, detected by sequencing all G235R, R236W and S264R lead to amino acid exchanges in the active coding parts of the whole genome, showed that it is possible that pa- site and, therefore, likely affect substrate binding and catalysis directly. tients with a completely different phenotype may have a pyrimidine The other three mutations L13S, R326Q and T359M did not affect resi- metabolism disturbance [30]. Recently, 4 asymptomatic neonates with dues located in or near the active site. The inactivity of the correspond- a putative ß-ureidopropionase deficiency were detected during a pilot ing mutant enzymes might be caused by folding defects and structural study of newborn urine screening and two infants during high risk instability. Additionally, all mutation sites are located near surfaces screening in Japan [31,32]. Thus, although these Japanese patients that are expected to become buried upon formation of higher oligomer- have not been confirmed molecularly, the identification of individuals ization states, and stabilization of lower molecular weight species has with biochemically complete ß-ureidopropionase deficiency without been observed for all further investigated mutant enzymes. Thus, anoth- any clinical manifestations and the marked intrafamilial variability in er explanation for the abolished or decreased enzymatic activity of L13S, phenotype, indicate that additional (epi)genetic and environmental R326Q and T359M may exist if ß-ureidopropionase is allosterically regu- factors are likely to be involved. lated by ligand-induced changes in oligomerization state as described for The pathogenesis underlying the various clinical manifestations re- the enzyme from rat liver and for some bacterial nitrilases [15,40–42].For mains as yet unknown. Biochemically, patients with a deficiency in these enzymes, association to larger oligomers was accompanied with in- one of the three enzymes of the pyrimidine degradation pathway creased activity, while the smallest assemblies (most likely the dimers) show moderately decreased levels of ß-alanine and strongly decreased were catalytically inactive. Analysis of the crystal structure of DmßUP levels of ß-aminoisobutyric acid [3,12,33]. Treatment of a patient with revealed a possible mechanism for such an allosteric regulation, involving ß-ureidopropionase deficiency with ß-alanine for over 1.5 years did the structural stabilization and fixation of three loops forming the en- not result in a clinical improvement [34]. ß-Aminoisobutyric acid is trance to and part of the active site upon interaction of dimeric units A.B.P. van Kuilenburg et al. / Biochimica et Biophysica Acta 1822 (2012) 1096–1108 1107

[18]. The catalytic inactivity of enzyme variants with point mutations that pyrimidine degradation associated with neurological abnormalities, Hum. Mol. Genet. 13 (2004) 2793–2801. prevent proper subunit association to higher oligomers would be in ac- [13] J. Yaplito-Lee, J. Pitt, J. Meijer, L. Zoetekouw, R. Meinsma, A.B.P. van Kuilenburg, cordance with such a model. Beta-ureidopropionase deficiency presenting with congenital anomalies of the The three enzymes of the pyrimidine degradation pathway are not urogenital and colorectal systems, Mol. Genet. Metab. 93 (2008) 190–194. [14] P. Vreken, A.B.P. van Kuilenburg, N. Hamajima, J.R. Meinsma, H. van Lenthe, G. only responsible for the catabolism of the naturally occurring bases Gohlich-Ratmann, B.E. Assmann, R.A. Wevers, A.H. van Gennip, cDNA cloning, ge- uracil and thymine, but also of the widely used chemotherapeutic nomic structure and chromosomal localization of the human BUP-1 gene encod- agent 5-fluorouracil. In this light, deficiencies of DPD and dihydropyr- ing beta-ureidopropionase, Biochim. Biophys. Acta 1447 (1999) 251–257. imidinase have been associated with severe toxicity after the administra- [15] M.M. Matthews, W. Liao, K.L. Kvalnes-Krick, T.W. Traut, Beta-alanine synthase: purification and allosteric properties, Arch. Biochem. Biophys. 293 (1992) 254–263. tion of 5-fluorouracil [43–45].Theidentification of a healthy individual [16] T. Sakamoto, S. Fujimoto Sakata, K. Matsuda, Y. Horikawa, N. Tamaki, Expression showing altered catabolism of uracil due to heterozygosity for a mutation and properties of human liver ß-ureidopropionase, J. Nutr. Sci. Vitaminol. 47 fi (2001) 132–138. in UPB1 suggests that also patients with a ß-ureidopropionase de ciency fi fl [17] G. Waldmann, P.F. Cook, K.D. Schnackerz, Puri cation and properties of beta- might be at risk of developing 5- uorouracil toxicity [46]. In addition, the alanine synthase from calf liver, Protein Pept. Lett. 12 (2005) 69–73. finding of 2 mutations in multiple ß-ureidopropionase patients of [18] S. Lundgren, B. Lohkamp, B. Andersen, J. Piskur, D. Dobritzsch, The crystal struc- different nationalities may indicate that a ß-ureidopropionase defi- ture of beta-alanine synthase from Drosophila melanogaster reveals a homooctameric helical turn-like assembly, J. Mol. Biol. 377 (2008) 1544–1559. ciency might not be as rare as generally considered. The prevalence of [19] Z. Gojkovic, M.P.B. Sandrini, J. Piskur, Eukaryotic beta-alanine synthases are func- a ß-ureidopropionase deficiency in Japan has been estimated to be 1 tionally related but have a high degree of structural diversity, Genetics 158 in 6000 [32]. The present results should facilitate the identification of (2001) 999–1011. fi [20] S. Lundgren, Z. Gojkovic, J. Piskur, D. 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