doi:10.1016/j.jmb.2008.05.

029

J. Mol. Biol. (2008) 380, 656666

Available online at www.sciencedirect.com

A Second Pathway to Degrade Pyrimidine Nucleic Acid Precursors in Eukaryotes

Gorm Andersen 1,2 , Olof Bjrnberg, Silvia Polakova 1 , Yuriy Pynyaha 1 , Anna Rasmussen 1 , Kasper Mller 2 , Anders Hofer 3 , Thomas Moritz 4 , Michael Paolo Bastner Sandrini 1,2 , Anna-Maria Merico 5 , Concetta Compagno 5 , Hans-Erik kerlund 6 , Zoran Gojkovi 2 and Jure Pikur 1,2
Department of Cell and Organism Biology, Lund University, 223 62 Lund, Sweden BioCentrum-DTU, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark Department of Medical Biochemistry and Biophysics, Ume University, 901 87 Ume, Sweden Ume Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 87 Ume, Sweden Department of Biomolecular Sciences and Biotechnology, University of Milan, 20133 Milan, Italy Department of Biochemistry, Center of Chemistry and Chemical Engineering, Lund University, 221 00 Lund, Sweden Received 14 January 2008; received in revised form 9 May 2008; accepted 10 May 2008 Available online 17 May 2008 Edited by J. Karn Keywords: 3-hydroxypropionic acid; metabolic pathways; nucleic acid precursors; uracil degradation; urea
6 5 4 3 2 1

Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC16 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. A combination of genetic and analytical chemistry methods demonstrates that uridine monophosphate and urea are intermediates, and 3-hydroxypropionic acid, ammonia and carbon dioxide the final products of degradation. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. The genes of the URC pathway are not homologous to any of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date.
2008 Elsevier Ltd. All rights reserved.

*Corresponding author. E-mail address: olof.bjornberg@cob.lu.se. G.A. and O.B. contributed equally to this work. Abbreviations used: BAL, -alanine; BUP, -ureidopropionic acid; BUPase, -ureidopropionase; DHU, dihydrouracil; DHPDHase, dihydropyrimidine dehydrogenase; DHPase, dihydropyrimidinase; EMS, ethyl methanesulfonate; GC-MS, gas chromatography-mass spectrometry; GTP, guanosine 5-triphosphate; ORF, open reading frame; SD, synthetic defined; TMS, trimethylsilyl; UPRTase, uracil phosphoribosyltransferase; URC, uracil catabolism; UMP, uridine monophosphate.
0022-2836/$ - see front matter 2008 Elsevier Ltd. All rights reserved.

Novel Yeast Metabolic Pathway

657 monophosphate (UMP) and urea are intermediates in the URC pathway, and the final outcome is 3hydroxypropionic acid and assimilated ammonia. The URC genes are widely distributed in fungi as well as in a variety of bacteria.

Introduction
In the living cell, nucleic acids are constantly built up and degraded. Biochemical pathways provide balanced pools of the precursors: bases, nucleosides and nucleotides.1 This metabolic network can be divided into de novo biosynthesis, salvage and degradation pathways. Pyrimidine bases have been considered to be degraded via either the reductive or the oxidative pathway.2 However, some organisms, such as baker's yeast, Saccharomyces cerevisiae, cannot degrade pyrimidines at all. The reductive pathway can be found in eukaryotes and some bacteria, and degrades uracil via dihydrouracil (DHU) and -ureidopropionic acid (BUP) to -alanine (BAL). The three reactions are catalyzed by dihydropyrimidine dehydrogenase (DHPDHase), dihydropyrimidinase (DHPase) and -ureidopropionase (BUPase), respectively.3 The reductive pathway is involved in human diseases4 and pharmacokinetics of pyrimidine-based anticancer drugs, such as 5-fluorouracil.5 The much less studied oxidative pathway is dependent on oxygen and has been found only in a few bacteria. Uracil is degraded via barbituric acid and ureidomalonic acid to urea and malonic acid,6 but so far only one enzyme of this pathway, barbiturase, has been characterized in detail.7 Escherichia coli K12 was for a long time wrongly considered to be unable to degrade uracil. Indeed, the E. coli genome does not contain genes for DHPDHase or barbiturase, which are needed for the reductive or oxidative uracil degradation, respectively, but recently, strains containing lesions in the b1012 operon, the largest cluster of uncharacterized E. coli genes, were shown to be unable to grow on uracil or uridine as sole N source.8 The deduced protein sequences of the operon (renamed as the rut operon) are involved in uracil degradation and show homology to several already characterized proteins, e.g., xanthine/uracil permease. The rut pathway needs an active respiratory chain and the carbon atoms corresponding to the uracil positions 4, 5 and 6 are secreted as 3-hydroxypropionic acid.8 We have intensively studied the yeast Saccharomyces kluyveri for its ability to grow on uracil as the sole N source. A genetic approach identified three loci, PYD2, PYD3 and PYD4, involved in the degradation of DHU to BUP, BUP to BAL, and BAL to malonic semialdehyde, respectively,911 and the structures and reaction mechanisms of the S. kluyveri DHPase12 and BUPase13 have recently been elucidated. However, a putative DHPDHase coding gene14 or the DHPDHase activity have not been identified in S. kluyveri. Homologs of the barbiturase gene or the rut genes cannot be found in the S. kluyveri genome either. In this work, we have used genetic, molecular biological and chemical approaches to show that in S. kluyveri, uracil is degraded by a novel pathway (URC), which is independent of the respiratory chain. The genes involved in the URC pathway have no homology to any of those participating in the known pyrimidine degradation pathways. Uridine

Results
PYD2 and PYD3 knockouts can use uracil as sole N source Previously, our laboratory described two genes, PYD2 and PYD3, as involved in the degradation of pyrimidines in S. kluyveri. The pyd2 (Y1019) and pyd3 (Y1021) strains, generated by ethyl methanesulfonate (EMS) mutagenesis, were unable to grow on DHU and BUP as sole N source, respectively.9,10 They were also both unable to grow on uracil. To further elucidate the role of these genes, two knockout strains, Y986 (pyd2-) and Y1046 (pyd3-), were constructed by directed gene disruption (Table 1). Both strains could grow on uracil but Y986 could not grow on DHU and Y1046 could not grow on either DHU or BUP as sole N source. This observation suggested that uracil degradation is independent of the PYD2 and PYD3 genes and that uracil and DHU are degraded by two different pathways. The previously described EMS mutants of PYD2 and PYD3 were presumably mutants in more than one locus. Uracil degradation is independent of oxygen S. kluyveri can, as well as S. cerevisiae, grow well in the absence of oxygen.15 Our practical definition of anaerobiosis (O2 b 3 ppm) excludes oxygendependent metabolism (e.g., the respiratory chain) but allows the oxygen-dependent enzyme ribonucleotide reductase to be active.16 The diploid reference strain Y057 (Table 1) grew well under aerobic and anaerobic conditions on different N sources (uracil, DHU and ammonia; Table 2). When the growth under aerobic and anaerobic conditions were compared, there was no large difference in the specific growth rates with uracil as sole N source (Table 2). Uracil degradation can therefore be concluded to be independent of oxygen. This conclusion was also supported by the fact that the specific growth rates and biomass yields under anaerobic conditions were similar with all three N sources. The three previously described degradation pathways are either oxygen dependent (the oxidative and rut pathways) or independent (the reductive one). The new pathway represents a second oxygen-independent pathway. Loci involved in uracil utilization S. kluyveri cells were mutagenized with EMS and mutants unable to utilize uracil as sole N source were isolated. In total, 45 urc (uracil catabolism) mutants were analyzed by complementation tests (see Supplementary Table S1). The mutants fell into

658
Table 1. Yeast strains used in this study
Designation Y057 Y090 Y091 Y156 Y159 Y786 Y787 Y804 Y805 Y806 Y807 Y808 Y810 Y811 Y813 Y814 Y815 Y816 Y817 Y842 Y843 Y844 Y845 Y846 Y847 Y848 Y849 Y850 Y852 Y853 Y855 Y856 Y857 Y935 Y936 Y937 Y948 Y950 Y951 Y952 Y953 Y954 Y957 Y958 Y959 Y960 Y961 Y962 Y963 Y964 Y986 Y1019 Y1021 Y1046 Y1156 Y1157 Y1158 Y1159 Reference/ origin NRRL Y-12651 L. Marsch, MYA-2152 L. Marsch, MYA-2153 J. Strathern, GRY1175 J. Strathern, GRY1183 Y159 Y156 Y159 Y159 Y159 Y156 Y156 Y156 Y156 Y156 Y159 Y159 Y156 Y156 Y156 Y156 Y159 Y156 Y159 Y159 Y156 Y159 Y159 Y159 Y159 Y159 Y156 Y159 Y156 Y156 Y156 Y159 Y159 Y159 Y159 Y159 Y159 Y159 Y159 Y159 Y156 Y159 Y159 Y159 Y159 Y156 Gojkovic et al.9 Gojkovic et al.10 Y156 Y90 Y90 Y91 Y91 Genotype Diploid, prototroph MAT thr MATa his aux MAT ura3 MATa ura3 MATa ura3 urc1 MAT ura3 urc2 MATa ura3 urc1 MATa ura3 urc2 MATa ura3 urc3 MAT ura3 urc5 MAT ura3 urc3 MAT ura3 urc4 MAT ura3 urc6 MAT ura3 urc4 MATa ura3 urc4 MATa ura3 urc4 MAT ura3 urc2 MAT ura3 urc2 MAT ura3 urc1 MAT ura3 urc5 MATa ura3 urc5 MAT ura3 urc4 MATa ura3 urc4 MATa ura3 urc4 MAT ura3 urc1 MATa ura3 urc3 MATa ura3 urc3 MATa ura3 urc3 MATa ura3 urc1 MATa ura3 urc1 MAT ura3 urc2 MATa ura3 urc3 MAT ura3 urc4 MAT ura3 urc4 MAT ura3 urc2 MATa ura3 urc4 MATa ura3 urc3 MATa ura3 urc3 MATa ura3 urc2 MATa ura3 urc3 MATa ura3 urc1 MATa ura3 urc1 MATa ura3 urc1 MATa ura3 urc3 MAT ura3 urc5 MATa ura3 urc3 MATa ura3 urc4 MATa ura3 urc3 MATa ura3 urc3 MAT ura3 pyd2KanMX3 MAT ura3 pyd2-1 MAT ura3 pyd3-1 MAT ura3 pyd3KanMX3 MAT thr urc1KanMX3 MAT thr urc1KanMX3 MATa his aux urc1KanMX3 MATa his aux urc2KanMX3 EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS Deletion EMS EMS Comments

Novel Yeast Metabolic Pathway Table 1 (continued)

Designation Y1160 Y1161 Y1162 Y1163 Y1165 Y1167 Y1168 Y1170 Y1172 Y1174 Y1212 Y1214 Reference/ origin Y91 Y156 Y156 Y90 Y91 Y156 Y90 Y91 Y90 Y91 Y90 Y91 Genotype MATa his aux urc2KanMX3 MAT ura3 urc2KanMX3 MAT ura3 urc2KanMX3 MAT thr urc3,5KanMX3 MATa his aux urc3,5KanMX3 MAT ura3 urc4KanMX3 MAT thr urc6KanMX3 MATa his aux urc6KanMX3 MAT thr urh1KanMX3 MATa his aux urh1KanMX3 MAT thr urk1KanMX3 MATa his aux urk1KanMX3 Comments Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion

aux stands for an unknown auxotrophic mutation.

six complementation groups termed urc1, urc2, urc3, urc4, urc5 and urc6. The individual genes were then identified by transformation and complementation with a S. kluyveri genomic library. The complementation groups, complementing plasmids and accession numbers for the genes are presented in Table 3. Each plasmid complements one specific urc mutation, except for the plasmid P637, which could complement both urc3 and urc5 mutants. Apparently, URC3 and URC5 belong to the same locus that we called URC3,5. Later on, strains carrying a disruption in each of the five URC genes were generated. The resulting deletion strains (urcx-) all failed to utilize uracil as sole N source, confirming the phenotypes of the EMS-induced mutants. Plasmid P540, which complemented the urc1 mutation, contained an open reading frame (ORF) termed URC1, encoding a protein with a putative guanosine 5-triphosphate (GTP) cyclohydrolase II motif. Urc1p showed low identity level in the C-terminal part (28% identical) to GTP cyclohydrolase II (YBL033Cp, Rib1p) from S. cerevisiae, but higher identity level to a group of putative cycloTable 2. Growth of S. kluyveri Y057 on media with different nitrogen sources, in the presence or absence of oxygen
Aerobic Anaerobic NH4+ 0.47 0.29 URA 0.21 0.07 DHU 0.24 0.07 NH4+ 0.24 0.09

Deletion Deletion Deletion Deletion Deletion (h ) Ysx (g/g)b

1 a

URA 0.25 0.26

DHU 0.24 0.23

URA, uracil; DHU, dihydrouracil. a , specific growth rate during the exponential growth on glucose. b Ysx, biomass yield grams dry weight biomass formed per gram glucose consumed (during the exponential growth on glucose).

Novel Yeast Metabolic Pathway Table 3. The urcx mutants obtained by EMS mutagenesis
Mutant locus urc1 urc2 urc3 urc4 urc5 urc6 Strains obtained 9 6 14 11 4 1 Complementing plasmid P540 P471 P637 P722 P637 P731 Accession no. AY154654 AY154653 DQ512718 DQ512719 DQ512718 DQ512720 S. cerevisae homolog (description)

659

None (cyclohydrolase ?) YDR520C (transcription factor) DUR1,2 (urea amidolyase) None (unknown) DUR1,2 (urea amidolyase) FUR1 (UPRTase*)

*Uracil phosphoribosyltransferase.

hydrolases found in fungi and bacteria with polypeptide chains about twice as long. Plasmid P722 complemented the urc4 mutants. It contained an ORF, termed URC4, encoding a protein that has no conserved domains. The two protein sequences of Urc1p and Urc4p were checked against the reference protein database (RefSeq), and the genes were found in several fungi and bacteria, but not in higher eukaryotes and archaea. Accession numbers for the homologous sequences found are presented in Table S2 in Supplementary Materials. A total of 34 species (13 fungi and 21 bacteria) were identified as having both Urc1p and Urc4p, while no organism having only one of the two was found. The remaining URC genes have homologs in S. cerevisiae (Table 3). Mutants of urc3 and urc5 could be complemented by the same plasmid carrying the complete gene for urea amidolyase. The encoded enzyme, with 1801 amino acid residues, is homologous (74% identity) to Dur1,2p of S. cerevisiae. The urc3,5 and the urc3,5- mutants were distinguished by their inability to utilize urea or allantoin as sole N sources, while the other urcx mutants could use both compounds. In contrast to the classical nickel-dependent urease found in, e.g., Schizosaccharomyces pombe, the enzymatic activity of urea amidolyase is bipartite. At one active site (Dur1p), ATP and urea form allophanate (urea carboxylate), which is hydrolyzed to ammonia and carbon dioxide at a separate active site, allophanate hydrolase (Dur2p).17 Apparently, the division of the enzyme gave rise to the two complementation groups, urc3 and urc5. The urc3,5 mutations strongly suggested that urea is an intermediate of uracil degradation. The Urc2p homolog in S. cerevisiae is the YDR520C gene product (52% identical), but the S. kluyveri gene contains two putative introns (536686 and 15611662 based on sequence homology). The S. cerevisiae gene has been connected to caffeine sensitivity18 and apparently encodes a zinc finger [Zn(2)Cys(6)] containing transcription factor, but does not have any other known function. BLAST homology search showed that URC2 is only found in 4 of the 18 annotated fungal species, namely, S. cerevisiae, Candida glabrata, Kluyveromyces lactis and Eremothecium gossypii. The URC6 gene encodes a protein that is homologous (87% identity) to S. cerevisiae uracil phosphoribosyltransferase (UPRTase) encoded by the FUR1 gene. The gene is widespread among microorganisms, and in some cases there is more than one copy

of the gene. All of the 34 organisms found to have URC1 and URC4 genes contain one or more URC6 homologous gene(s). Accession numbers for Urc6p sequences found in these organisms are also included in Table S2 in the Supplementary Materials. URC1, URC4 and URC6 are often linked The genomic locations of the URCX genes in several sequenced genomes were determined. In almost all of the bacteria [Synechoccus sp. JA-2-3B'a (213) is the only exception] having URC1 and URC4 homologs, these genes were either located next to each other or as overlapping loci, indicating a polycistronic organization (e.g., Bradyrhizobium japonicum, Fig. 1a). In all 20 cases, a putative UPP gene, the bacterial homolog of FUR1, was found downstream of URC1 and URC4. Furthermore, in Bdellovibrio bacteriovorus HD100, the cluster of URC1, URC4 and URC6 is flanked by two other genes of interest, putatively encoding methylmalonate semialdehyde dehydrogenase (mmsA, NP_968421) and uridine kinase (udk, NP_968417), respectively. It is interesting to note that although Bradyrhizobium sp. BTAi1 has a cluster of URC1, URC4 and URC6, it also has a cluster of genes [pydX (YP_001241873), pydA (YP_001241872), pydB (YP_001241866) and pydC (YP_001241865)] encoding the components of an apparently intact reductive pathway for degradation of uracil. In eukaryotes, it is relatively rare to see the genes belonging to the same pathway located next to each other, but this is the case with the URC1 and URC4 genes in S. pombe (Fig. 1b). They are flanked on one side by a bacteria-like UPRTase gene (UPP) and on the other side by three genes encoding a putative Zn (2)Cys(6) protein, a yeast-like UPRTase (FUR1) and a yeast-like uracil transporter (FUR4), respectively. The transcript levels of the three neighboring genes in S. pombe, called Urg13 (uracil-regulatable genes, corresponding to URC 1, URC4 and UPP in Fig. 1b), are strongly increased in response to uracil.19 In Neurospora crassa and Yarrowia lipolytica, UPP (URC6) is found together with URC1 and URC4, respectively (Fig. 1c and d). However, in S. kluyveri there is no clustering of the URC genes. UPRTase is necessary for degradation Only one EMS mutant, Y811, was isolated within the urc6 complementation group and its uracil phe-

660

Novel Yeast Metabolic Pathway

Fig. 1. Organization of the genes homologous to S. kluyveri URC1, URC4 and URC6/FUR1 in some eubacteria (a) and fungi (bd). (a) B. japonicum (NC_004463, region: 79570907963265), (b) S. pombe (NC_003424, region: 1831000..1844000), (c) N. crassa (NW_047266, region: Comp(68218..83644)), (d) Y. lipolytica (CR382131, region: 2440600..2446600). In bacteria (a), URC1 and URC4 are located either as closely spaced or overlapping loci. Next to them is a putative UPP (bacterial type UPRTase), which is homologous to URC6/FUR1. In the yeast S. pombe (b), both a UPP and a yeast URC6/FUR1 homolog are located nearby. The URC6/FUR1 gene is flanked by a FUR4 (uracil transporter) homolog and a gene encoding a putative Zn(2)Cys(6) motif protein. In the yeasts N. crassa and Y. lipolytica, UPP (URC6) is located together with either URC1 or URC4, respectively (c and d).

notype was rescued by FUR1 encoding UPRTase. Unlike the other 44 EMS mutants, Y811 could grow on uridine. The relevance of the complementation group was not obvious because the strains used for EMS mutagenesis, Y156 and Y159, are ura3 and thus dependent on the salvage of uracil through UPRTase. Y811 did not show increased resistance to 5-fluorouracil, whose toxicity is mediated by UPRTase, and approximately 60% of the wild-type UPRTase activity was observed for cells grown in YPD medium (data not shown). Apparently, Y811 carried a leaky mutation. However, the knockout strains (urc6-), generated in the URA3 background provided a clear result. These knockout strains, Y1168 and Y1170, were unable to degrade uracil and were highly resistant to 5-fluorouracil (5 mM). Inactivation of URH1 and URK1 To further investigate the role of pyrimidine salvage, we inactivated two other genes, URH1 and URK1, encoding uridine hydrolase and uridine kinase, respectively. Like FUR1, they have counterparts in S. cerevisiae that have been thoroughly annotated,2022 and here the only known route from uridine to uracil is via uridine hydrolase. Our diploid urh1 deletion mutant in S. kluyveri (Y1172Y1174) showed similar growth as the parental strain (Y090Y091) on uridine as sole N source (data not shown). We analyzed the activity of extracts from the knockout strain Y1172 (urh1-) in phosphate buffer to confirm that this strain does not contain hydrolases/phosphorylases to cleave uridine. No conversion of uridine to uracil was detected in extracts from

the knockout strain (Fig. 2), which excludes the possibility that uracil is the first committed substrate in the degradation pathway. By the sequential action of uridine hydrolase and UPRTase, a strain with a disrupted URK1 gene can convert uridine to UMP. Our diploid urk1 deletion

Fig. 2. Uridine hydrolase is absent in Y1172 (urh1-). The uridine hydrolase/phosphorylase activity in a urh1 knockout strain was compared to the activity present in the parental strain, Y090. The two reaction mixtures contained crude protein extract (1 mg/ml) from Y1172 (urh1-) (filled circles) and Y090 (URH1) (open circles) and uridine (0.5 mM) in phosphate buffer. Aliquots were removed from the mixture and treated and analyzed for remaining uridine on a C18 column.

Novel Yeast Metabolic Pathway

661

mutant (Y1212Y1214) grew well on uracil but only slowly on uridine (Fig. 3). This quantitative phenotype during growth on uridine indicates that phosphorylation of uridine to UMP rather than hydrolysis to uracil is a preferred route. The urk1 deletion mutants showed another phenotype, resistance to 5-fluorouridine (1 mM), very similar to that found for the corresponding knockouts in S. cerevisiae.22,23 The properties of the urc6, urh1 and urk1 deletion mutants suggested that UMP is a mandatory intermediate of the URC pathway. Excreted degradation product(s) of uracil
14

C-labeled

We used the diploid reference strain Y057 to follow the depletion of 14C-labeled uracil in minimal medium with uracil as sole N source. Label in the C2 position disappeared rapidly, while label in the C6 position stayed constant during the whole growth period except for a transient drop in the exponential phase (Fig. 4). The loss of label in the C2 position is consistent with formation of urea, a substrate of urea amidolyase (Urc3,5p), and subsequent release of the C2 atom as CO2. The N1 and N3 atoms liberated as ammonium ions are conceivably assimilated during growth. However, the retaining of label in the C6 position suggested that the C6 atom returned to the media as a part of one or more excretion products.

Fig. 4. Depletion of radioactivity from the supernatant of S. kluyveri Y057 grown in the presence of labeled uracil. The cells were grown for 72 h with 1 mM uracil as sole N source, labeled with either [14C2]- or [14C6]uracil (empty and filled triangles, respectively). OD600 (empty circles) is also shown. The remaining label in the supernatant is shown as disintegrations per minute per microliter.

Identification of 3-hydroxypropionic acid in the medium Y057 cells were grown in a mixture (1:1) of unlabeled uracil and [13C4,5]uracil. Subsequent analysis of the medium by gas chromatographymass spectrometry (GCMS) identified unlabeled and 13 C-labeled trimethylsilyl (TMS)-derivatized 3hydroxypropionic acid with molecular ions at m/z 219 and 221, respectively. Thus, the MS spectrum (Fig. 5) indicated that the compound indeed originated from uracil. Furthermore, when medium from Y057 cells grown on [14C6]uracil was analyzed by HPLC (as described in Materials and Methods), radioactivity eluted in one broad peak at 11 2 min (data not shown). A corresponding sample of 13 C4,5-labeled material was chromatographed, and in the collected fractions, only 3-hydroxypropionic acid could be identified by GCMS. Urea is an intermediate of uracil degradation The urea amidolyase-deficient Y852 strain (urc3) was grown in the presence of 14C-labeled uracil, and labeled products in cell extracts and the growth medium were analyzed by HPLC. A predominant 14 C-labeled peak with a retention time of 6.0 min was observed from both the cell extract (the low molecular weight fraction obtained by perchloric acid extraction) and medium from cells incubated with [ 14 C2]uracil but not from cells incubated with [14C6]uracil. The retention time of this peak was the same as for 14C-labeled urea. When nonmutagenized (parental) Y159 cells were incubated

Fig. 3. Growth phenotype of the urk1 deletion mutant. Deletion of URK1 encoding uridine kinase was performed in both Y090 and Y091, giving the new strains Y1212 and Y1214, respectively. The two strains were mated and the growth properties of the resulting diploid (urk1) were tested. As a reference, the corresponding diploid (URK1) from a mating of Y090 and Y091 was used. A total of 10,000 cells in 2 l (OD = 0.1) were spotted on uridine and uracil and the growth was recorded after 3 days. The urk1 mutant grows slower on uridine than the corresponding parental strain.

662

Novel Yeast Metabolic Pathway

Fig. 5. Identification of TMS-derivatized 3-hydroxypropionic acid as the excreted product from uracil degradation. S. kluyveri cells (Y057) were grown to stationary phase in an equal mixture of unlabeled uracil and [13C4,5]uracil as sole N source and cell-free supernatant was used for GCMS analysis. The molecular ions at m/z 219 and 221 demonstrate that the compound is derived from uracil. The reference spectrum for TMS-derivatized 3-hydroxypropionic acid is shown in the bottom part.

in a corresponding experiment with [14C2]uracil, only a small peak was detected. From the parallel incubations of Y852 (urc3) and Y159 cells with [14C6]uracil, nearly all radioactivity, both in the cell extract and medium, appeared to be present as either 3-hydroxypropionic acid or unmetabolized uracil. To make the medium amenable to conclusive GC MS analysis we switched to 13C2-labeled uracil. The medium from Y852 cells was derivatized with TMS and analyzed. A compound that eluted after 215 s had a mass spectrum matching TMS-derivatized [13C]urea, with a molecular weight of m/z 205 compared to m/z 204 for unlabeled TMSurea standard (Fig. 6). [M 15]+ ions were observed at m/z 190 for the [13C]urea and m/z 189 for unlabeled standard confirming the genetic data that urea is generated from uracil.

Discussion
For a long time, pyrimidine bases were thought to be degraded by either a reductive pathway via DHU or an oxidative pathway via barbituric acid.24 However, Loh et al. recently described a third pathway (rut), which requires the presence of oxygen and operates in E. coli K12 and in some other proteobacteria.8 Hereby we can report the fourth pathway, URC, operating in a wide range of yeast, fungi and bacteria. It is the second reported pathway for degradation in eukaryotes.

The yeast S. kluyveri can use uracil and all intermediates of the reductive pyrimidine catabolic pathway as sole N sources,25 which we first interpreted as uracil degradation by the reductive pathway.9,10 When we knocked out the PYD2 or PYD3 genes, encoding DHPase and BUPase, respectively, the resulting pyd2- and pyd3- strains were surprisingly still able to grow on uracil as sole N source. Thus, uracil is not degraded via DHU in S. kluyveri. The growth experiments demonstrated that the new degradation pathway, URC (for uracil catabolism), can operate also anaerobically (Table 2) and is thus different from the oxidative and rut pathways. Forty-five EMS-induced mutants unable to grow on uracil but still capable of using ammonia were mapped to six complementation groups (Table 3 and Table S1). Two of them, urc3 and 5, represented two parts of the same gene (URC3,5) coding for urea amidolyase. The other four complementation groups consisted of two genes, URC2 and URC6, having identified homologs in S. cerevisiae, and URC1 and URC4, coding for proteins with so far unknown functions. The well-known protein UPRTase (encoded by URC6), converting uracil to UMP, is necessary for growth on uracil. During growth on uridine, however, UPRTase is bypassed, and uridine kinase is used to supply the UMP. It appears expensive to use UMP in comparison to the reductive and oxidative pathways of degradation, in which the uracil molecule is employed as a substrate of DHPDHase or uracil oxidase to yield DHU or barbituric acid, respectively.

Novel Yeast Metabolic Pathway

663

Fig. 6. Identification of urea from Y852 cells grown with 13C2-labeled uracil. The MS spectrum shown on the top graph is taken from a GC peak that eluted at 215 s. The bottom graph shows a reference GCMS spectrum from unlabeled urea. Both samples were derivatized with TMS prior to analysis.

Like in the URC pathway, urea is generated in the bacterial oxidative pathway and could therefore have been indicative of an oxidative mechanism in the yeast S. kluyveri. However, degradation is not dependent on oxygen and none of the identified URC genes shares any homology to the only identified gene encoding barbiturase7 in the oxidative pathway. Neither do the URC genes show any similarity to the E. coli rut genes, although the end product, 3-hydroxypropionic acid, is the same. Urea is not a likely intermediate of the rut pathway, since E. coli K12 lacks urease and urea amidolyase. The URC1 and URC4 homologs can be found in a number of fungi and bacteria (Supplementary Material, Table S2). It is likely that URC1 and URC4 are the core components of the pathway because their homologs (together with the UPRTase-encoding gene) are linked in a majority of the analyzed bacterial genomes (Fig. 1a). Co-localization is also seen in yeast, e.g., S. pombe (Fig. 1b). It is noteworthy that Urc1p has a domain with a putative GTP cyclohydrolase II motif. By analogy, the first substrate of the URC pathway may be uridine triphosphate. It is possible to speculate upon the function of Urc1p and the unknown intermediates on the basis of the sequence homology to GTP cyclohydrolase II, the identified intermediate, urea, and the waste product 3-hydroxypropionic acid (Fig. 7). The uracil ring could be opened hydrolytically at C6, in a manner similar to that of the opening of the guanine ring at the C8 position by GTP cyclohydrolase II.26 The C6 of uracil sits between the N1 and C5 atoms, whereas C8 in the guanine ring is between two N atoms and

conceivably more easy to attack hydrolytically. Further hydrolytic removal of the ribose and hydrolysis at C4 would release urea and malonic semialdehyde. Only urea was conclusively identified in this study, while malonic semialdehyde may be converted by a hypothetical aldehyde reductase to the detected waste product, 3-hydroxypropionic acid. One should keep in mind that our genetic screen may not have picked up all genes required for uracil degradation, especially if they are essential for the

Fig. 7. URC-based degradation of uracil. The N1C2 N3 atoms of the uracil molecule are released as urea that is further degraded by Urc3,5p (ATP-dependent urea amidolyase) to ammonia and carbon dioxide. The C4C5C6 carbon atoms end up as the waste product 3-hydroxypropionic acid. The gene products of URC1 and URC4 are unknown proteins, while URC2 encodes a putative transcription factor that also is essential for degradation. The URC6 gene product, UPRTase, furnishes UMP, which is a mandatory intermediate. When uridine is used as N-source, the role of URC6 is replaced by the gene encoding uridine kinase (URK1).

664 general metabolism, i.e., required for growth on ammonia as N source. In conclusion, S. kluyveri and many other fungi and bacteria use a hitherto unknown pyrimidine degradation pathway. The URC pathway may have been present in the last common progenitor of bacteria and eukaryotes but later lost in several prokaryote and eukaryote lineages.

Novel Yeast Metabolic Pathway onto new SD or uracil N-minimal plates. After 57 days at 25 C, putative mutant colonies were chosen based on their inability to grow on uracil N-minimal plates. Strains were grouped based on the interallelic complementation tests, which were carried out by crossing mutants on YPD plates and replica-plating to uracil N-minimal plates (see Supplementary Materials, Table S1). Complementation of urc mutants

Materials and Methods

Materials Uracil, 5-fluorouracil, DHU, BUP and BAL were purchased from Sigma. Yeast nitrogen base without amino acids and ammonium sulfate was purchased from Difco. [14C2]Uracil (MC124), [14C6]uracil (MC159), [14C2]uridine (MC105) and [14C]urea (MC141) were purchased from Moravek Biochemicals (Brea, CA). [13 C2]Uracil and [13C4,5]uracil were purchased from Sigma and Cambridge Isotope, Inc., respectively. Oligos used were from DNA Technology (Aarhus, Denmark) and sequencing was done by MWG Biotech (Ebersberg, Germany). Strains and growth media The S. kluyveri strains used for the knockout and mutagenesis studies and the strains produced in these studies are presented in Table S1. The strains were grown in YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose) or synthetic defined (SD)/N-minimal medium (1% succinic acid, 0.6% sodium hydroxide, 2% glucose, 0.17% yeast nitrogen base without amino acids and ammonium sulfate) supplemented with different N sources [0.5% ammonium sulfate (SD), and 0.1% for other N sources unless otherwise stated]. For solid medium (plates), 2% agar was added. The E. coli strain XL1-Blue (Stratagene) was used for plasmid amplification. Bacteria were grown at 37 C in LuriaBertani medium supplemented with 100 mg/l of ampicillin for selection.27 G418 selection media consisted of YPD supplemented with 75 mg/l of G418 (Sigma G5013). Fermentor experiments Batch cultivations were performed at 30 C in 2-l jacketed bioreactors (Applikon, Schiedam, the Netherlands), with a working volume of 1.0 l. The aerobic cultures were aerated with 1.0 l air per minute, and the anaerobic cultures were sparged with pure nitrogen (b 3 ppm oxygen) at a flow rate of 0.2 l N2 per minute. The dissolved oxygen tension was measured with an autoclavable O2 sensor (Mettler Toledo), and it was at all times during the cultivations above 20% of air saturation for the aerated cultures and zero for the nitrogen-sparged cultures. Cultivations were performed in glucose minimal medium as previously described15 with either uracil, DHU or ammonium sulfate as N source. For anaerobic cultivations, the bioreactor was flushed with nitrogen for at least 24 h prior to inoculation. Mutagenesis Yeast mutants were generated from the strains Y156 and Y159 with EMS as described.28 Mutagenized cells were plated on YPD plates (100200 colonies per plate) and grown for 23 days at 25 C. The plates were replicated The S. kluyveri wild type genomic library prepared by F. Lacroute was based on the shuttle vector pFL44S.29 Mutants from each of the complementation groups were transformed with the library DNA by electroporation9 and plated on uracil N-minimal plates for selection. A number of transformants from each mutant strain were tested for plasmid loss, before rescue of the plasmid into the E. coli strain. Sequencing of the inserts was done using the primers M13rev-29 and M13uni-21 from MWG Biotech. The complementation groups and the rescued plasmids along with accession numbers for the complementing ORFs are presented in Table 3. Sequence analysis Nucleotide sequence analysis and protein alignments were done with WinSeqEZ ver. 1.0 (F. G. Hansen, unpublished data) and ClustalX ver. 1.8. Database searches were performed using the default setup at the BLAST network services at the National Center for Biotechnology Information. Gene disruptions Replacement cassettes with flanking homologous regions (approximately 500 bp) were used to disrupt the PYD2, PYD3 and URCX genes. These homology regions, 5 and 3 parts of the gene, were designed to remove the start codon and at least two-thirds of the targeted ORF. PCR amplification was performed with Pfu polymerase (Stratagene) from wild-type genomic DNA (strain Y057). All oligos used are presented in Supplementary Materials (Table S3). Our gene-specific primers were produced (1-5, 1-3, 2-5 and 2-3) in order to amplify two 500-bp fragments of each gene to be knocked out. The 1-3 and 2-5 primers contained 25-bp extensions homologous to the 1.5-kb kanMX3 cassette, which confers geneticin (G418) resistance. The cassette was amplified from the plasmid pFA6-kanMX3.30 In a second PCR amplification, the two 500-bp fragments were mixed with the kanMX3 cassette, and a PCR product consisting of the cassette flanked by the two 500-bp fragments was produced using the 1-5 and 2-3 primers. The resulting linear fragments of each 2500 bp were used to transform cells using electroporation as described9 and selected on G418 plates. Correct integration of these inserts was confirmed by PCR. Deletions were done in Y156, or Y090 and Y091. The uracil phenotype was tested directly in the Y156 background (ura3). Strains with URC deletions obtained in both Y090 (MAT thr) and Y091 (MATa his aux) were crossed to obtain prototrophic diploids, homozygous with respect to the deletion, for tests of the uracil phenotype.

www.ncbi.nlm.nih.gov/BLAST/

Novel Yeast Metabolic Pathway Assays of uridine hydrolase/phosphorylase activity Crude extracts of Y1172 (urh1-) and Y090 (wild type) cells grown in YPD medium were obtained by 10 cycles of freezing (in liquid nitrogen) and thawing in 50 mM potassium phosphate buffer (pH 7.5), 5 mM dithiothreitol and protease inhibitors (Complete, from Roche Diagnostics). After centrifugation, the extract was passed through a gel-filtration column (PD-10). The reaction mixture (3.0 ml) contained 3.0 mg of protein in 50 mM potassium phosphate (pH 7.5). The reaction, at 25 C, was started by the addition of uridine to 0.5 mM. Aliquots of 200 l were removed and mixed with an equal volume of 10% trichloric acid. The precipitate was removed by centrifugation and 350 l of the supernatant was neutralized with 100 l of 1.0 M NaOH and further diluted with 200 l H2O. Treated reaction mixture (100 l) was loaded on a VYDAC RP C18 column run isocratically with 2% methanol and 10 mM ammonium acetate (pH 5.0) at 1 ml/min to separate uridine and uracil (conditions kindly provided by Paul Klein, Middle Tennessee State University, Murfreesboro, TN, personal communication, 2006). The remaining amount of uridine was calculated from its peak area. Uptake of uracil Uptake of uracil was followed in 5-ml cultures of strain Y057 with uracil (1 mM) as sole N source. The cultures were labeled with 0.5 Ci of either [14C2]uracil or [14C6] uracil. Samples were withdrawn at time points up to 72 h, cells were removed by centrifugation, and the radioactivity in the supernatant was determined by liquid scintillation counting. Analysis of medium and intracellular extracts by HPLC Y159 (wild type) and Y852 (urc3) cells were grown in proline (0.1%)/uracil (0.1%) N-minimal media. Y852 cannot grow on uracil, but it was assumed that the enzymes of the pathway were induced by its presence. Cells were harvested at OD600 = 1.0 (approximately 5 107 cells/ml), washed and concentrated in SD media without ammonium sulfate. The following incubation with 14C2- or 14C6labeled uracil was performed in this medium in a volume of 300 l including uracil to a final concentration of 0.7 mM (1.8 Ci/mol) and allowed to continue for 90 min. Cells and medium were separated by centrifugation and collected. Cells were resuspended in 250 l perchloric acid (1.0 M) and incubated on ice for 10 min. After centrifugation, the supernatant was neutralized with KOH and the precipitate formed was removed by centrifugation. Samples were analyzed on a 100 mm 4.6 mm 200 5 ZIC-pHILIC column (SeQuant, Ume, Sweden), run at 1 ml/min, with a mobile phase of 90% acetonitrile/2 mM ammonium carbonate (made from a 20 mM stock solution of pH 9.6). The peaks were detected by a UV spectrophotometer (260 nm) and a continuous liquid scintillation counter (Packard Flo-One). This chromatographic system, suitable for subsequent GCMS analysis, was initially designed to separate uracil, uridine and 14C-labeled urea. By lowering the content of acetonitrile to 85% or 80%, also phosphorylated nucleosides could be analyzed. Very little label was found as nucleotides and no label was found at retention times corresponding to those of some 14C-labeled reference compounds from other uracil degradation pathways (BUP, BAL and barbituric acid). GCMS analysis

665

Prior to GCMS analysis, an aliquot of the sample was dried, and 30 l of methoxyamine hydrochloride (15 mg/ ml) in pyridine was added. After 16 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h at room temperature by adding 30 l of N-methyl-Ntrimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (Pierce, Rockford, IL). After silylation, 30 l of heptane was added. One microliter of the derivatized sample was injected splitless by an Agilent 7683 autosampler (Agilent, Atlanta, GA) into an Agilent 6890 gas chromatograph following a procedure that has been described previously.31 All data were processed by ChromaTOF (2.12) software (Leco Corp., St. Joseph, MI). Automatic peak detection and mass spectrum deconvolution were performed using a peak width set to 1.5 s. The detected compounds were identified by comparing retention indices and mass spectra with data in retention index and mass spectra libraries.32

Acknowledgements
We thank Lise Schack at the Department of Biological Chemistry at Copenhagen University for performing assays on the UPRTase activity. We also thank Klaus Schnackerz for his interest in this project. This work was supported by grants from the Danish and Swedish Research Councils, the Swedish Cancer Society, and the Crafoord and Srensen Foundations.

Supplementary Data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2008.05.029

References
1. Mathews, C. K., van Holde, K. E. & Ahern, K. G. (2000). Nucleotide metabolism. In Biochemistry, 3rd edit., pp. 794829. Benjamin/Cummings Publishing Company, San Francisco, CA. 2. Vogels, G. D. & van der Drift, C. (1976). Degradation of purines and pyrimidines by microorganisms. Bacteriol. Rev. 40, 403468. 3. Wasternack, C. (1980). Degradation of pyrimidines and pyrimidine analogspathways and mutual influences. Pharmacol. Ther. 8, 629651. 4. van Gennip, A. H., Abeling, N. G., Vreken, P. & van Kuilenburg, A. B. (1997). Inborn errors of pyrimidine degradation: clinical, biochemical and molecular aspects. J. Inherit. Metab. Dis. 20, 203213. 5. Kubota, T. (2003). 5-fluorouracil and dihydropyrimidine dehydrogenase. Int. J. Clin. Oncol. 8, 127131. 6. Hayaishi, O. & Kornberg, A. (1952). Metabolism of cytosine, thymine, uracil, and barbituric acid by bacterial enzymes. J. Biol. Chem. 197, 717732. 7. Soong, C. L., Ogawa, J., Sakuradani, E. & Shimizu, S. (2002). Barbiturase, a novel zinc-containing amidohy-

666
drolase involved in oxidative pyrimidine metabolism. J. Biol. Chem. 277, 70517058. Loh, K. D., Gyaneshwar, P., Papadimitriou, E. M., Fong, R., Kim, K.-S., Parales, R. et al. (2006). A previously undescribed pathway for pyrimidine catabolism. Proc. Natl. Acad. Sci. USA, 103, 51145119. Gojkovic, Z., Jahnke, K., Schnackerz, K. D. & Pikur, J. (2000). PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in a novel fungal catabolic pathway. J. Mol. Biol. 295, 10731087. Gojkovic, Z., Sandrini, M. P. & Pikur, J. (2001). Eukaryotic -alanine synthases are functionally related but have a high degree of structural diversity. Genetics, 158, 9991011. Andersen, G., Andersen, B., Dobritzsch, D., Schnackerz, K. D. & Pikur, J. (2007). A gene duplication led to specialized -aminobutyrate and -alanine aminotransferase in yeast. FEBS J. 274, 18041817. Lohkamp, B., Andersen, B., Pikur, J. & Dobritzsch, D. (2006). The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity. J. Biol. Chem. 281, 1376213776. Lundgren, S., Gojkovic, Z., Pikur, J. & Dobritzsch, D. (2003). Yeast -alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases. J. Biol. Chem. 278, 5185151862. Cliften, P., Sudarsanam, P., Desikan, A., Fulton, L., Fulton, B., Majors, J. et al. (2003). Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science, 301, 7176. Moller, K., Olsson, L. & Piskur, J. (2001). Ability for anaerobic growth is not sufficient for development of the petite phenotype in Saccharomyces kluyveri. J. Bacteriol. 183, 24852489. Nordlund, P. & Reichard, P. (2006). Ribonucleotide reductases. Annu. Rev. Biochem. 75, 681706. Cooper, T. G., Lam, C. & Turoscy, V. (1980). Structural analysis of the dur loci in S. cerevisiae: two domains of a single multifunctional gene. Genetics, 94, 555580. Akache, B., Wu, K. & Turcotte, B. (2001). Phenotypic analysis of genes encoding yeast zinc cluster proteins. Nucleic Acids Res. 29, 21812190. Watt, S., Mata, J., Lopez-Maury, L., Marguerat, S., Burns, G. & Bahler, J. (2008). urg1: a uracil-regulatable promoter system for fission yeast with short induction and repression times. PLoS ONE, 3, e1428. Kern, L. (1990). The URK1 gene of Saccharomyces

Novel Yeast Metabolic Pathway cerevisiae encoding uridine kinase. Nucleic Acids Res. 18, 5279. Mitterbauer, R., Karl, T. & Adam, G. (2002). Saccharomyces cerevisiae URH1 (encoding uridinecytidine N-ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase. Appl. Environ. Microbiol. 68, 13361343. Kurtz, J. E., Exinger, F., Erbs, P. & Jund, R. (2002). The URH1 uridine ribohydrolase of Saccharomyces cerevisiae. Curr. Genet. 41, 132141. Kurtz, J. E., Exinger, F., Erbs, P. & Jund, R. (1999). New insights into the pyrimidine salvage pathway of Saccharomyces cerevisiae: requirement of six genes for cytidine metabolism. Curr. Genet. 36, 130136. Pikur, J., Schnackerz, K. D., Andersen, B. & Bjrnberg, O. (2007). Comparative genomics points out novel biochemical pathways. Trends Genet. 23, 369372. LaRue, T. A. & Spencer, J. F. (1968). The utilization of purines and pyrimidines by yeasts. Can. J. Microbiol. 14, 7986. Ren, R., Kotaka, M., Lockyer, M., Lamb, H. K. & Hawkins, A. R. (2005). GTP cyclohydrolase II structure and mechanism. J. Biol. Chem. 280, 3691236919. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. Gojkovic, Z., Paracchini, S. & Piskur, J. (1998). A new model organism for studying the catabolism of pyrimidines and purines. Adv. Exp. Med. Biol. 431, 475479. Bonneaud, N., Ozier-Kalogeropoulos, O., Li, G. Y., Labouesse, M., Minvielle-Sebastia, L. & Lacroute, F. (1991). A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast, 7, 609615. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. (1994). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast, 10, 17931808. Gullberg, J., Jonsson, P., Nordstrm, A., Sjstrm, M. & Moritz, T. (2004). Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry. Anal. Biochem. 331, 283295. Schauer, N., Steinhauser, D., Strelkov, S., Schomburg, D., Allison, G., Moritz, T. et al. (2005). GCMS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett. 579, 13321337.

8.

21.

9.

22. 23.

10.

11.

24. 25. 26. 27. 28. 29.

12.

13.

14.

15.

16. 17.

30.

31.

18. 19.

32.

20.