a second pathway to degrade pyrimidine nucleic acid precursors in eukary

8/13/2019 A Second Pathway to Degrade Pyrimidine Nucleic Acid Precursors in Eukary

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A Second Pathway to Degrade Pyrimidine Nucleic AcidPrecursors in Eukaryotes

Gorm Andersen1,2, Olof Bjrnberg, Silvia Polakova1,Yuriy Pynyaha1, Anna Rasmussen1, Kasper Mller2, Anders Hofer3,Thomas Moritz4, Michael Paolo Bastner Sandrini1,2,Anna-Maria Merico5, Concetta Compagno5, Hans-Erik kerlund6,Zoran Gojkovi2 and Jure Pikur1,2

1Department of Cell andOrganism Biology, LundUniversity, 223 62 Lund, Sweden2BioCentrum-DTU, TechnicalUniversity of Denmark, 2800Kgs. Lyngby, Denmark3Department of MedicalBiochemistry and Biophysics,Ume University, 901 87Ume, Sweden4Ume Plant Science Center,

Department of Forest Geneticsand Plant Physiology, SwedishUniversity of AgriculturalSciences, 901 87 Ume, Sweden5Department of BiomolecularSciences and Biotechnology,University of Milan, 20133Milan, Italy6Department of Biochemistry,Center of Chemistry andChemical Engineering, LundUniversity, 221 00 Lund,

SwedenReceived 14 January 2008;received in revised form9 May 2008;accepted 10 May 2008Available online17 May 2008

Pyrimidine bases are the central precursors for RNA and DNA, and theirintracellular pools are determined by de novo, salvage and catabolicpathways. In eukaryotes, degradation of uracil has been believed toproceed only via the reduction to dihydrouracil. Using a yeast model,Saccharomyces kluyveri, we show that during degradation, uracil is notreduced to dihydrouracil. Six loci, named URC16(for uracil catabolism),are involved in the novel catabolic pathway. Four of them, URC3,5,URC6,andURC2encode urea amidolyase, uracil phosphoribosyltransferase, and aputative transcription factor, respectively. The gene products ofURC1andURC4 are highly conserved proteins with so far unknown functions andthey are present in a variety of prokaryotes and fungi. In bacteria and insome fungi, URC1and URC4are linked on the genome together with the

gene for uracil phosphoribosyltransferase (URC6

). Urc1p and Urc4p aretherefore likely the core components of this novel biochemical pathway. Acombination of genetic and analytical chemistry methods demonstrates thaturidine monophosphate and urea are intermediates, and 3-hydroxypro-pionic acid, ammonia and carbon dioxide the final products of degradation.The URC pathway does not require the presence of an active respiratorychain and is therefore different from the oxidative and rut pathwaysdescribed in prokaryotes, although the latter also gives 3-hydroxypropionicacid as the end product. The genes of the URC pathway are not homologousto any of the eukaryotic or prokaryotic genes involved in pyrimidinedegradation described to date.

2008 Elsevier Ltd. All rights reserved.

Edited by J. KarnKeywords: 3-hydroxypropionic acid; metabolic pathways; nucleic acidprecursors; uracil degradation; urea

*Corresponding author.E-mail address:[email protected]. 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.

doi:10.1016/j.jmb.2008.05.029 J. Mol. Biol.(2008)380, 656666

Available online at www.sciencedirect.com

0022-2836/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
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Introduction

In the living cell, nucleic acids are constantly builtup and degraded. Biochemical pathways provide

balanced pools ofthe precursors: bases, nucleosidesand nucleotides.1 This metabolic network can bedivided intode novobiosynthesis, salvage and degra-dation pathways. Pyrimidine bases have been con-sidered to be degraded via either the reductive or theoxidative pathway.2 However, some organisms, suchas baker's yeast, Saccharomyces cerevisiae, cannot de-grade pyrimidines at all.

The reductive pathway can be found in eukaryotesand some bacteria, and degrades uracil via dihydro-uracil (DHU) and -ureidopropionic acid (BUP) to-alanine (BAL). The three reactions are catalyzed bydihydropyrimidine dehydrogenase (DHPDHase),dihydropyrimidinase (DHPase) and -ureidopro-

pionase (BUPase), respectively.3

Thereductive path-way is involved in human diseases4 and pharmaco-kinetics of pyrimidine-based anticancer drugs, suchas 5-fluorouracil.5

The much less studied oxidative pathway is de-pendent on oxygen and has been found only in a few

bacteria. Uracil is degraded via barbituric acid andureidomalonic acid to urea and malonic acid,6but sofar only one enzyme of this pathway, barbiturase,has been characterized in detail.7

Escherichia coli K12 was for a long time wronglyconsidered to be unable to degrade uracil. Indeed, theE. coli genome does not contain genes for DHPDHaseor barbiturase, which are needed for the reductive

or oxidative uracil degradation, respectively, butrecently, strains containing lesions in the b1012operon, the largest cluster of uncharacterizedE. coligenes, were shown to be unable to grow on uracil oruridine as sole N source.8 The deduced protein se-quences of the operon (renamed as the rutoperon)are involved in uracil degradation and show homo-logy to several already characterized proteins, e.g.,xanthine/uracil permease. Therutpathway needs anactive respiratory chain and the carbon atoms corres-ponding to the uracil positions 4, 5 and 6 are secretedas 3-hydroxypropionic acid.8

We have intensively studied the yeast Saccharo-

myces kluyverifor its ability to grow on uracil as thesole N source. A genetic approach identified threeloci, PYD2, PYD3 and PYD4, involved in the de-gradation of DHU to BUP, BUP to BAL,and BALto malonic semialdehyde, respectively,911 and thestructuresand reactionmechanisms of theS. kluyveriDHPase12 and BUPase13 have recently been eluci-dated. However, a putative DHPDHase coding gene14or the DHPDHase activity have not been identified inS. kluyveri. Homologs of the barbiturase gene or the rutgenes cannot be found in the S. kluyveri genome either.

In this work, we have used genetic, molecularbiological and chemical approaches to show that inS. kluyveri, uracil is degraded by a novel pathway

(URC), which is independent of the respiratorychain. The genes involved in the URC pathway haveno homology to any of those participating in theknown pyrimidine degradation pathways. Uridine

monophosphate (UMP) and urea are intermediatesin the URC pathway, and the final outcome is 3-hydroxypropionic acid and assimilated ammonia.The URCgenes are widely distributed in fungi aswell as in a variety of bacteria.

Results

PYD2and PYD3knockouts can use uracil assole N source

Previously, our laboratory described two genes,PYD2and PYD3, as involved in the degradation ofpyrimidines in S. kluyveri. The pyd2 (Y1019) andpyd3 (Y1021) strains, generated by ethyl methane-sulfonate (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 (Table1). Both strains could grow on uracil but Y986 couldnot grow on DHU and Y1046 could not grow oneither DHU or BUP as sole N source. This observationsuggested that uracil degradation is independent ofthePYD2andPYD3genes and that uracil and DHUare degraded by two different pathways. The pre-viously described EMS mutants ofPYD2and PYD3were presumably mutants in more than one locus.

Uracil degradation is independent of oxygen

S. kluyverican, as wellas S. cerevisiae, grow wellin the absence of oxygen.15 Our practical definitionof anaerobiosis (O2 b3 ppm) excludes oxygen-dependent metabolism (e.g., the respiratory chain)

but allows the oxygen-dependent enzyme ribonu-cleotide reductase to be active.16 The diploid refe-rence strain Y057 (Table 1) grew well under aerobicand anaerobic conditions on different N sources(uracil, DHU and ammonia; Table 2). When thegrowth under aerobic andanaerobic conditions werecompared, there was no large difference in the spe-

cific growth rates with uracil as sole N source (Table2). Uracil degradation can therefore be concluded tobe independent of oxygen. This conclusion was alsosupported by the fact that the specific growth ratesand biomass yields under anaerobic conditions weresimilarwith all three N sources. The three previouslydescribed degradation pathways are either oxygendependent (the oxidative andrutpathways) or inde-pendent (the reductive one). The new pathway re-presents a second oxygen-independent pathway.

Loci involved in uracil utilization

S. kluyvericells were mutagenized with EMS and

mutants unable to utilize uracil as sole N sourcewere isolated. In total, 45 urc (uracil catabolism)mutants were analyzed by complementation tests(see Supplementary Table S1). The mutants fell into

657Novel Yeast Metabolic Pathway


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six complementation groups termedurc1,urc2,urc3,urc4,urc5andurc6. The individual genes were thenidentified by transformation and complementationwith aS. kluyverigenomic library. The complemen-tation groups, complementing plasmids and acces-sion numbers for the genes are presented inTable 3.

Each plasmid complements one specific urcmuta-tion, except for the plasmid P637, which could com-plement both urc3 and urc5 mutants. Apparently,URC3and URC5belong to the same locus that wecalled URC3,5. Later on, strains carrying a disrup-tion in each of the five URC genes were generated.The resulting deletion strains (urcx-) all failed toutilize uracil as sole N source, confirming the pheno-types of the EMS-induced mutants.

Plasmid P540, which complemented the urc1mu-tation, contained an open reading frame (ORF)termed URC1, encoding a protein with a putativeguanosine 5-triphosphate (GTP) cyclohydrolase IImotif. Urc1p showed low identity level in theC-terminal part (28% identical) to GTP cyclohy-drolase II (YBL033Cp, Rib1p) fromS. cerevisiae, buthigher identity level to a group of putative cyclo-

Table 2. GrowthofS. kluyveri Y057 on media with differentnitrogen sources, in the presence or absence of oxygen

Aerobic Anaerobic

URA DHU NH4+ URA DHU NH4

+

(h1)a 0.25 0.24 0.47 0.21 0.24 0.24Ysx(g/g)

b 0.26 0.23 0.29 0.07 0.07 0.09

URA, uracil; DHU, dihydrouracil.a , specific growth rate during the exponential growth on

glucose.b Ysx, biomass yieldgrams dry weight biomass formed pergram

glucose consumed (during the exponential growth on glucose).

Table 1.Yeast strains used in this study

DesignationReference/

origin Genotype Comments

Y057 NRRL Y-12651 Diploid,

prototrophY090 L. Marsch,MYA-2152

MATthr

Y091 L. Marsch,MYA-2153

MATahis aux

Y156 J. Strathern,GRY1175

MATura3

Y159 J. Strathern,GRY1183

MATaura3

Y786 Y159 MATaura3 urc1 EMSY787 Y156 MATura3 urc2 EMSY804 Y159 MATaura3 urc1 EMSY805 Y159 MATaura3 urc2 EMSY806 Y159 MATaura3 urc3 EMSY807 Y156 MATura3 urc5 EMSY808 Y156 MATura3 urc3 EMSY810 Y156 MATura3 urc4 EMSY811 Y156 MATura3 urc6 EMSY813 Y156 MATura3 urc4 EMSY814 Y159 MATaura3 urc4 EMSY815 Y159 MATaura3 urc4 EMSY816 Y156 MATura3 urc2 EMSY817 Y156 MATura3 urc2 EMSY842 Y156 MATura3 urc1 EMSY843 Y156 MATura3 urc5 EMSY844 Y159 MATaura3 urc5 EMSY845 Y156 MATura3 urc4 EMSY846 Y159 MATaura3 urc4 EMSY847 Y159 MATaura3 urc4 EMSY848 Y156 MATura3 urc1 EMSY849 Y159 MATaura3 urc3 EMSY850 Y159 MATaura3 urc3 EMSY852 Y159 MATaura3 urc3 EMS

Y853 Y159 MATa

ura3 urc1 EMSY855 Y159 MATaura3 urc1 EMSY856 Y156 MATura3 urc2 EMSY857 Y159 MATaura3 urc3 EMSY935 Y156 MATura3 urc4 EMSY936 Y156 MATura3 urc4 EMSY937 Y156 MATura3 urc2 EMSY948 Y159 MATaura3 urc4 EMSY950 Y159 MATaura3 urc3 EMSY951 Y159 MATaura3 urc3 EMSY952 Y159 MATaura3 urc2 EMSY953 Y159 MATaura3 urc3 EMSY954 Y159 MATaura3 urc1 EMSY957 Y159 MATaura3 urc1 EMSY958 Y159 MATaura3 urc1 EMSY959 Y159 MATaura3 urc3 EMSY960 Y156 MATura3 urc5 EMS

Y961 Y159 MATaura3 urc3 EMSY962 Y159 MATaura3 urc4 EMSY963 Y159 MATaura3 urc3 EMSY964 Y159 MATaura3 urc3 EMSY986 Y156 MATura3

pyd2KanMX3Deletion

Y1019 Gojkovicet al.9

MATura3pyd2-1

EMS

Y1021 Gojkovicet al.10

MATura3pyd3-1

EMS

Y1046 Y156 MATura3pyd3KanMX3

Deletion

Y1156 Y90 MATthrurc1KanMX3

Deletion


Deletion

Y1158 Y91 MATahis auxurc1KanMX3

Deletion


Deletion

Table 1(continued)

DesignationReference/

origin Genotype Comments


Deletion

Y1161 Y156 MATura3urc2KanMX3

Deletion


Deletion

Y1163 Y90 MATthrurc3,5KanMX3

Deletion

Y1165 Y91 MATahis auxurc3,5KanMX3

Deletion


Deletion


Deletion


Deletion

Y1172 Y90 MATthrurh1KanMX3

Deletion

Y1174 Y91 MATahis auxurh1KanMX3 DeletionY1212 Y90 MATthr

urk1KanMX3Deletion

Y1214 Y91 MATahis auxurk1KanMX3

Deletion

auxstands for an unknown auxotrophic mutation.

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hydrolases found in fungi and bacteria with poly-peptide chains about twice as long. Plasmid P722complemented the urc4 mutants. It contained anORF, termedURC4, encoding a protein that has noconserved domains. The two protein sequences of

Urc1p and Urc4p were checked against the referenceprotein database (RefSeq), and the genes were foundin several fungi and bacteria, but not in highereukaryotes and archaea. Accession numbers for thehomologous sequences found are presented in TableS2 in Supplementary Materials. A total of 34 species(13 fungi and 21 bacteria) were identified as having

both Urc1p and Urc4p, while no organism havingonly one of the two was found.

The remaining URC genes have homologs inS. cerevisiae (Table 3). Mutants of urc3 and urc5could be complemented by the same plasmid carry-ing the complete gene for urea amidolyase. Theencoded enzyme, with 1801 amino acid residues, is

homologous (74% identity) to Dur1,2p ofS. cerevisiae.The urc3,5 and the urc3,5- mutants were distin-guished by their inability to utilize urea or allan-toin as sole N sources, while the other urcxmutantscould use both compounds. In contrast to the classicalnickel-dependent urease found in, e.g., Schizosaccha-romyces pombe, the enzymatic activity of urea amido-lyase is bipartite. At one active site (Dur1p), ATP andurea form allophanate (urea carboxylate), which ishydrolyzed to ammonia and carbon dioxide at aseparate active site, allophanate hydrolase (Dur2p).17Apparently, the division of the enzyme gave rise tothe two complementation groups,urc3andurc5. The

urc3,5mutations strongly suggested that urea is anintermediate of uracil degradation.The Urc2p homolog in S. cerevisiaeis theYDR520C

gene product (52% identical), but theS. kluyverigenecontains two putative introns (536686 and 15611662

based on sequence homology). The S. cerevisiaegenehas been connected to caffeine sensitivity18 and appa-rently encodes a zinc finger [Zn(2)Cys(6)] containingtranscription factor, but does not have any otherknown function. BLAST homology search showedthat URC2 is only found in 4 of the 18 annotatedfungal species, namely, S. cerevisiae, Candida glabrata,Kluyveromyces lactisandEremothecium gossypii.

The URC6 gene encodes a protein that is homo-

logous (87% identity) toS. cerevisiaeuracil phospho-ribosyltransferase (UPRTase) encoded by the FUR1gene. The gene is widespread among microorga-nisms, and in some cases there is more than one copy

of the gene. All of the 34 organisms found to haveURC1and URC4genes contain one or more URC6homologous gene(s). Accession numbers for Urc6psequences found in these organisms are also includedin Table S2 in the Supplementary Materials.

URC1, URC4and URC6are often linked

The genomic locations of the URCX genes inseveral sequenced genomes were determined. Inalmost all of the bacteria [Synechoccus sp. JA-2-3B'a(213) is the only exception] having URC1andURC4homologs, these genes were either located next toeach other or as overlapping loci, indicating a poly-cistronic organization (e.g.,Bradyrhizobium japonicum,Fig. 1a). In all 20 cases, a putative UPPgene, the

bacterial homolog ofFUR1, was found downstreamof URC1 and URC4. Furthermore, in BdellovibriobacteriovorusHD100, the cluster ofURC1,URC4andURC6is flanked by two other genes of interest, puta-tively encoding methylmalonate semialdehyde dehy-drogenase (mmsA, NP_968421) and uridine kinase(udk, NP_968417), respectively. It is interesting to notethat althoughBradyrhizobiumsp. BTAi1 has a clusterofURC1, URC4 and URC6, it also has a cluster ofgenes [pydX(YP_001241873), pydA(YP_001241872),pydB (YP_001241866) and pydC (YP_001241865)]encoding the components of an apparently intactreductive pathway for degradation of uracil.

In eukaryotes, it is relatively rare to see the genesbelonging to the same pathway located next to eachother, but this is the case with theURC1andURC4

genes inS. pombe(Fig. 1b). They are flanked on oneside by a bacteria-like UPRTase gene (UPP) and onthe other side by three genes encoding a putative Zn(2)Cys(6) protein, a yeast-like UPRTase (FUR1)andayeast-like uracil transporter (FUR4), respectively.The transcript levels of the three neighboring genesinS. pombe, calledUrg13(uracil-regulatable genes,corresponding toURC 1,URC4andUPPinFig. 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. kluyverithere is no clustering of the URCgenes.

UPRTase is necessary for degradation

Only one EMS mutant, Y811, was isolated withintheurc6complementation group and its uracil phe-

Table 3.The urcxmutants obtained by EMS mutagenesis

Mutantlocus

Strainsobtained

Complementingplasmid Accession no.

S. cerevisaehomolog(description)

urc1 9 P540 AY154654 None (cyclohydrolase ?)

urc2 6 P471 AY154653 YDR520C(transcription factor)urc3 14 P637 DQ512718 DUR1,2(urea amidolyase)urc4 11 P722 DQ512719 None (unknown)urc5 4 P637 DQ512718 DUR1,2(urea amidolyase)urc6 1 P731 DQ512720 FUR1(UPRTase*)

*Uracil phosphoribosyltransferase.



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notype was rescued by FUR1 encoding UPRTase.Unlike the other 44 EMS mutants, Y811 could growon uridine. The relevance of the complementation

group was not obvious because the strains usedfor EMS mutagenesis, Y156 and Y159, are ura3andthus dependent on the salvage of uracil throughUPRTase. Y811 did not show increased resistanceto 5-fluorouracil, whose toxicity is mediated byUPRTase, and approximately 60% of the wild-typeUPRTase activity was observed for cells grown inYPD medium (data not shown). Apparently, Y811carried a leaky mutation. However, the knockoutstrains (urc6-), generated in theURA3backgroundprovided a clear result. These knockout strains,Y1168 and Y1170, were unable to degrade uracil andwere highly resistant to 5-fluorouracil (5 mM).

Inactivation of URH1and URK1

To further investigate the role of pyrimidine sal-vage, we inactivated two other genes, URH1 andURK1, encoding uridine hydrolase and uridinekinase, respectively. Like FUR1, they have counter-parts in S. cerevisiae that have been thoroughlyannotated,2022 and here the only known route fromuridine to uracil is via uridine hydrolase. Our diploidurh1 deletion mutant in S. kluyveri (Y1172Y1174)showed similar growth as the parental strain(Y090Y091) on uridine as sole N source (data notshown). We analyzed the activity of extracts from the

knockout strain Y1172 (urh1-) in phosphate bufferto confirm that this strain does not contain hydro-lases/phosphorylases to cleave uridine. No conver-sion of uridine to uracil was detected in extracts from

the knockout strain (Fig. 2), which excludes the pos-sibility that uracil is the first committed substrate inthe degradation pathway.

By the sequential action of uridine hydrolase andUPRTase, a strain with a disrupted URK1gene canconvert uridine to UMP. Our diploid urk1deletion

Fig. 1. Organization of the genes homologous toS. kluyveri URC1,URC4andURC6/FUR1in some eubacteria (a) andfungi (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 typeUPRTase), which is homologous toURC6/FUR1. In the yeastS. pombe(b), both a UPPand a yeastURC6/FUR1homologare located nearby. The URC6/FUR1gene is flanked by a FUR4(uracil transporter) homolog and a gene encoding aputative Zn(2)Cys(6) motif protein. In the yeasts N. crassaand Y. lipolytica, UPP (URC6) is located together with eitherURC1or URC4, respectively (c and d).

Fig. 2. Uridine hydrolase is absent in Y1172 (urh1-).The uridine hydrolase/phosphorylase activity in a urh1knockout strain was compared to the activity present inthe parental strain, Y090. The two reaction mixturescontained crude protein extract (1 mg/ml) from Y1172(urh1-) (filled circles) and Y090 (URH1) (open circles) anduridine (0.5 mM) in phosphate buffer. Aliquots wereremoved from the mixture and treated and analyzed forremaining uridine on a C18column.



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mutant (Y1212Y1214) grew well on uracil but onlyslowly on uridine (Fig. 3). This quantitative pheno-type during growth on uridine indicates thatphosphorylation of uridine to UMP rather thanhydrolysis to uracil is a preferred route. The urk1deletion mutants showed another phenotype, resis-tance to 5-fluorouridine (1 mM), very similar to thatfound for the corresponding knockouts in S.cerevisiae.22,23 The properties of the urc6, urh1 andurk1 deletion mutants suggested that UMP is amandatory intermediate of the URC pathway.

Excreted degradation product(s) of 14C-labeleduracil

We used the diploid reference strain Y057 tofollow the depletion of14C-labeled uracil in minimalmedium with uracil as sole N source. Label in the C2

position disappeared rapidly, while label in the C6position stayed constant during the whole growthperiod except for a transient drop in the exponentialphase (Fig. 4). The loss of label in the C2position isconsistent with formation of urea, a substrate ofurea amidolyase (Urc3,5p), and subsequent releaseof the C2atom as CO2. The N1and N3atoms libe-rated as ammonium ions are conceivably assimi-lated during growth. However, the retaining oflabel in the C6position suggested that the C6atomreturned to the media as a part of one or more ex-cretion products. Identification of 3-hydroxypropionic acid in the

medium

Y057 cells were grown in a mixture (1:1) of un-labeleduracil and [13C4,5]uracil. Subsequent analysisof the medium by gas chromatographymassspectrometry (GCMS) identified unlabeled and13C-labeled trimethylsilyl (TMS)-derivatized 3-hydroxypropionic acid with molecular ions at m/z219 and 221, respectively. Thus, the MS spectrum(Fig. 5) indicated that the compound indeed ori-ginated from uracil. Furthermore, when mediumfrom 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 of13

C4,5-labeled material was chromatographed, andin the collected fractions, only 3-hydroxypropionicacid could be identified by GCMS.

Urea is an intermediate of uracil degradation

The urea amidolyase-deficient Y852 strain (urc3)was grown in the presence of14C-labeled uracil, andlabeled products in cell extracts and the growthmedium were analyzed by HPLC. A predominant14C-labeled peak with a retention time of 6.0 minwas observed from both the cell extract (the lowmolecular weight fraction obtained by perchloricacid extraction) and medium from cells incubated

with [14

C2]uracil but not from cells incubatedwith [14C6]uracil. The retention time of this peakwas the same as for 14C-labeled urea. When non-mutagenized (parental) Y159 cells were incubated

Fig. 3. Growth phenotype of theurk1deletion mutant.Deletion ofURK1encoding uridine kinase was performedin both Y090 and Y091, giving the new strains Y1212 andY1214, respectively. The two strains were mated and thegrowth properties of the resulting diploid (urk1) weretested. As a reference, the corresponding diploid (URK1)from a mating of Y090 and Y091 was used. A total of10,000 cells in 2 l (OD=0.1) were spotted on uridine anduracil and the growth was recorded after 3 days. Theurk1- mutant grows slower on uridine than the correspondingparental strain.

Fig. 4. Depletion of radioactivity from the supernatantofS. kluyveriY057 grown in the presence of labeled uracil.The cells were grown for 72 h with 1 mM uracil as sole Nsource, labeled with either [14C2]- or [

14C6]uracil (emptyand filled triangles, respectively). OD600(empty circles) isalso shown. The remaining label in the supernatant isshown as disintegrations per minute per microliter.



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in a corresponding experiment with [14C2]uracil,

only a small peak was detected.From the parallel incubations of Y852 (urc3) andY159 cells with [14C6]uracil, nearly all radioactivity,

both in the cell extract and medium, appeared to bepresent as either 3-hydroxypropionic acid or unme-tabolized uracil.

To make the medium amenable to conclusive GCMS analysis we switched to 13C2-labeled uracil. Themedium from Y852 cells was derivatized with TMSand analyzed. A compound that eluted after 215 shad a mass spectrum matching TMS-derivatized[13C]urea, with a molecular weight ofm/z205 com-pared tom/z204 for unlabeled TMSurea standard

(Fig. 6). [M

15]

+

ions were observed atm/z190 forthe [13C]urea and m/z 189 for unlabeled standardconfirming the genetic data that urea is generatedfrom uracil.

Discussion

For a long time, pyrimidine bases were thought tobe degraded by either a reductive pathway via DHUor an oxidative pathway via barbituric acid.24 How-ever, Loh et al.recently described a third pathway(rut), which requires the presence of oxygen andoperates in E. coli K12 and in some other proteo-

bacteria.8

Hereby we can report the fourth pathway,URC, operating in a wide range of yeast, fungi andbacteria. It is the second reported pathway fordegradation in eukaryotes.

The yeast S. kluyveri can use uracil and all inter-

mediates of the reductivepyrimidine catabolic path-way as sole N sources,25 which we first interpretedas uracil degradation by the reductive pathway.9,10

When we knocked out the PYD2 or PYD3 genes,encoding DHPase and BUPase, respectively, theresulting pyd2- and pyd3- strains were surpris-ingly 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 newdegradation pathway, URC (for uracil catabolism),can operate also anaerobically (Table 2) and is thusdifferent from the oxidative and rutpathways.

Forty-five EMS-induced mutants unable to grow

on uracil but still capable of using ammonia weremapped to six complementation groups (Table 3and Table S1). Two of them, urc3 and 5, repre-sented two parts of the same gene (URC3,5) codingfor urea amidolyase. The other four complemen-tation groups consisted of two genes, URC2 andURC6, having identified homologs in S. cerevisiae,and URC1 and URC4, coding for proteins with so farunknown functions.

The well-known protein UPRTase (encoded byURC6), converting uracil to UMP, is necessary forgrowth on uracil. During growth on uridine, how-ever, UPRTase is bypassed, and uridine kinase is usedto supply the UMP. It appears expensive to use UMP

in comparison to the reductive and oxidative path-ways of degradation, in which the uracil molecule isemployed as a substrate of DHPDHase or uraciloxidase to yield DHU or barbituric acid, respectively.

Fig. 5. Identification of TMS-derivatized 3-hydroxypropionic acid as the excreted product from uracil degradation.S. kluyvericells (Y057) were grown to stationary phase in an equal mixture of unlabeled uracil and [13C4,5]uracil as sole Nsource and cell-free supernatant was used for GCMS analysis. The molecular ions at m/z219 and 221 demonstrate thatthe compound is derived from uracil. The reference spectrum for TMS-derivatized 3-hydroxypropionic acid is shown inthe bottom part.



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Like in the URC pathway, urea is generated in thebacterial oxidative pathway and could therefore

have been indicative of an oxidative mechanism inthe yeast S. kluyveri. However, degradation is notdependent on oxygen and none of the identifiedURCgenes shares any homology to the only iden-tified gene encoding barbiturase7 in the oxidativepathway. Neither do theURCgenes show any simi-larity to theE. coli rutgenes, although the end pro-duct, 3-hydroxypropionic acid, is the same. Urea isnot a likely intermediate of the rutpathway, sinceE. coliK12 lacks urease and urea amidolyase.

TheURC1andURC4homologs can be found in anumber of fungi and bacteria (SupplementaryMaterial, Table S2). It is likely thatURC1andURC4

are the core componentsof the pathway becausetheir homologs (together with the UPRTase-encodinggene) are linked in a majority of the analyzed bac-terial genomes (Fig.1a). Co-localization is also seen inyeast, e.g., S. pombe (Fig. 1b). It is noteworthy thatUrc1p has a domain with a putative GTP cyclo-hydrolase II motif. By analogy, the first substrate ofthe URC pathway may be uridine triphosphate. It ispossible to speculate upon the function of Urc1pand the unknown intermediates on the basis of thesequence homology to GTP cyclohydrolase II, theidentified intermediate, urea, and the waste product3-hydroxypropionic acid (Fig. 7). The uracil ringcould be opened hydrolytically at C6, in a manner

similar to that of the opening of the guanine ring atthe C8position by GTP cyclohydrolase II.26 The C6ofuracil sits between the N1and C5atoms, whereas C8in the guanine ring is between two N atoms and

conceivably more easy to attack hydrolytically. Fur-ther hydrolytic removal of the ribose and hydrolysis

at C4would release urea and malonic semialdehyde.Only urea was conclusively identified in this study,while malonic semialdehyde may be converted by ahypothetical aldehyde reductase to the detectedwaste product, 3-hydroxypropionic acid.

One should keep in mind that our genetic screenmay not have picked up all genes required for uracildegradation, especially if they are essential for the

Fig. 6. Identification of urea from Y852 cells grown with 13C2-labeled uracil. The MS spectrum shown on the top graphis 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.

Fig. 7. URC-based degradation of uracil. The N1C2N3atoms of the uracil molecule are released as urea that isfurther degraded by Urc3,5p (ATP-dependent urea ami-dolyase) to ammonia and carbon dioxide. The C4C5C6carbon atoms end up as the waste product 3-hydroxypro-pionic acid. The gene products of URC1 and URC4 areunknown proteins, while URC2 encodes a putativetranscription factor that also is essential for degradation.TheURC6gene product, UPRTase, furnishes UMP, whichis a mandatory intermediate. When uridine is used asN-source, the role of URC6 is replaced by the geneencoding uridine kinase (URK1).



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general metabolism, i.e., required for growth onammonia as N source. In conclusion, S. kluyveriandmany other fungi and bacteria use a hitherto un-known pyrimidine degradation pathway. The URCpathway may have been present in the last commonprogenitor of bacteria and eukaryotes but later lostin several prokaryote and eukaryote lineages.

Materials and Methods

Materials

Uracil, 5-fluorouracil, DHU, BUP and BAL were pur-chased from Sigma. Yeast nitrogen base without aminoacids and ammonium sulfate was purchased from Difco.[14C2]Uracil (MC124), [

14C6]uracil (MC159), [14C2]uridine

(MC105) and [14C]urea (MC141) were purchased fromMoravek Biochemicals (Brea, CA). [13C2]Uracil and[13C4,5]uracil were purchased from Sigma and CambridgeIsotope, Inc., respectively. Oligos used were from DNATechnology (Aarhus, Denmark) and sequencing was done

by MWG Biotech (Ebersberg, Germany).

Strains and growth media

TheS. kluyveristrains used for the knockout and muta-genesis studies and the strains produced in these studies arepresented in Table S1. The strains were grown in YPDmedium (1% yeast extract, 2% bactopeptone, 2% glucose) orsynthetic defined (SD)/N-minimal medium (1% succinicacid, 0.6% sodium hydroxide, 2% glucose, 0.17% yeastnitrogen base without amino acids and ammonium sulfate)

supplemented with different N sources [0.5% ammoniumsulfate (SD), and 0.1% for other N sources unless otherwisestated]. For solid medium (plates), 2% agar was added. TheE. coli strain XL1-Blue (Stratagene) was used for plasmidamplification. Bacteria were grown at 37 C in LuriaBertanimedium supplemented with 100 mg/l of ampicillin forselection.27 G418 selection media consisted of YPD supple-mented with 75 mg/l of G418 (Sigma G5013).

Fermentor experiments

Batch cultivations were performed at 30 C in 2-ljacketed bioreactors (Applikon, Schiedam, the Nether-lands), with a working volume of 1.0 l. The aerobic

cultures were aerated with 1.0 l air per minute, and theanaerobic cultures were sparged with pure nitrogen(b3 ppm oxygen) at a flow rate of 0.2 l N2 per minute.The dissolved oxygen tension was measured with anautoclavable O2sensor (Mettler Toledo), and it was at alltimes during the cultivations above 20% of air saturationfor the aerated cultures and zero for the nitrogen-spargedcultures. Cultivations were performed in glucose minimalmedium as previously described15 with either uracil, DHUor ammonium sulfate as N source. For anaerobic cultiva-tions, the bioreactor was flushed with nitrogen for at least24 h prior to inoculation.

Mutagenesis

Yeast mutants were generated from the strains Y156 andY159 with EMS as described.28 Mutagenized cells wereplated on YPD plates (100200 colonies per plate) andgrown for 23 days at 25 C. The plates were replicated

onto new SD or uracil N-minimal plates. After 57 days at25 C, putative mutant colonies were chosen based ontheir inability to grow on uracil N-minimal plates. Strainswere grouped based on the interallelic complementationtests, which were carried out by crossing mutants on YPD

plates and replica-plating to uracil N-minimal plates (seeSupplementary Materials, Table S1).

Complementation of urcmutants

The S. kluyveriwild type genomic library prepared byF. Lacroute was based on the shuttle vector pFL44S.29Mutants from each of the complementation groups weretransformed with the library DNA by electroporation9

and plated on uracil N-minimal plates for selection. Anumber of transformants from each mutant strain weretested for plasmid loss, before rescue of the plasmid intotheE. colistrain. Sequencing of the inserts was done usingthe primers M13rev-29 and M13uni-21 from MWG

Biotech. The complementation groups and the rescuedplasmids along with accession numbers for the comple-menting ORFs are presented inTable 3.

Sequence analysis

Nucleotide sequence analysis and protein alignmentswere done with WinSeqEZ ver. 1.0 (F. G. Hansen,unpublished data) and ClustalX ver. 1.8. Databasesearches were performed using the default setup at theBLAST network services at the National Center forBiotechnology Information.

Gene disruptions

Replacement cassettes with flanking homologousregions (approximately 500 bp) were used to disrupt thePYD2,PYD3and URCXgenes. These homology regions,5and 3parts of the gene, were designed to remove thestart 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, 2-5and 2-3) in order to amplify two 500-bp frag-ments of each gene to be knocked out. The 1-3and 2-5primers contained 25-bp extensions homologous to the1.5-kb kanMX3 cassette, which confers geneticin (G418)resistance. Thecassette was amplified from the plasmidpFA6-kanMX3.30 In a second PCR amplification, thetwo 500-bp fragments were mixed with the kanMX3cassette, and a PCR product consisting of the cassetteflanked by the two 500-bp fragments was produced usingthe 1-5and 2-3primers. The resulting linear fragmentsof each 2500 bp were used to transform cells using elec-troporation as described9 and selected on G418 plates.Correct integration of these inserts was confirmed byPCR. Deletions were done in Y156, or Y090 and Y091.The uracil phenotype was tested directly in the Y156

background (ura3). Strains with URCdeletions obtainedin both Y090 (MAT thr) and Y091 (MATa his aux)were crossed to obtain prototrophic diploids, homozy-gous with respect to the deletion, for tests of the uracilphenotype.

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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 offreezing (in liquid nitrogen) and thawing in 50 mM

potassium phosphate buffer (pH 7.5), 5 mM dithiothreitoland protease inhibitors (Complete, from Roche Diagnos-tics). After centrifugation, the extract was passed througha gel-filtration column (PD-10). The reaction mixture(3.0 ml) contained 3.0 mg of protein in 50 mM potassiumphosphate (pH 7.5). The reaction, at 25 C, was started

by the addition of uridine to 0.5 mM. Aliquots of 200 lwere removed and mixed with an equal volume of 10%trichloric acid. The precipitate was removed by centri-fugationand 350l of thesupernatantwas neutralizedwith100 l of 1.0 M NaOH and further diluted with 200 l H2O.Treated reaction mixture (100 l) was loaded on a VYDACRP C18 column run isocratically with 2% methanol and10 mM ammonium acetate (pH 5.0) at 1 ml/min to separateuridine and uracil (conditions kindly provided by Paul

Klein, Middle Tennessee State University, Murfreesboro,TN, personal communication, 2006). The remainingamount of uridine was calculated from its peak area.

Uptake of uracil

Uptake of uracil was followed in 5-ml cultures of strainY057 with uracil (1 mM) as sole N source. The cultureswere 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 radio-activity in the supernatant was determined by liquid scin-tillation counting.

Analysis of medium and intracellular extracts byHPLC

Y159 (wild type) and Y852 (urc3) cells were grown inproline (0.1%)/uracil (0.1%) N-minimal media. Y852 cannotgrow on uracil, but it was assumed that the enzymes ofthe pathway were induced by its presence. Cells wereharvested at OD600=1.0 (approximately 510

7 cells/ml),washed and concentrated in SD media without ammo-nium sulfate. The following incubation with 14C2- or 14C6-labeled uracil was performed in this medium in a volumeof 300 l including uracil to a final concentration of0.7 mM (1.8 Ci/mol) and allowed to continue for 90 min.

Cells and medium were separated by centrifugationand collected. Cells were resuspended in 250l perchloricacid (1.0 M) and incubated on ice for 10 min. After cen-trifugation, the supernatant was neutralized with KOHand the precipitate formed was removed by centrifuga-tion. Samples were analyzed on a 100 mm4.6 mm 200 5ZIC-pHILIC column (SeQuant, Ume, Sweden), run at1 ml/min, with a mobile phase of 90% acetonitrile/2 mMammonium carbonate (made from a 20 mM stock solutionof pH 9.6). The peaks were detected by a UV spectro-photometer (260 nm) and a continuous liquid scintillationcounter (Packard Flo-One).

This chromatographic system, suitable for subsequentGCMS analysis, was initially designed to separate uracil,uridine and 14C-labeled urea. By lowering the content ofacetonitrile to 85% or 80%, also phosphorylated nucleo-sides could be analyzed. Very little label was found asnucleotides and no label was found at retention timescorresponding to those of some 14C-labeled referencecompounds from other uracil degradation pathways(BUP, BAL and barbituric acid).

GCMS analysis

Prior to GCMS analysis, an aliquot of the sample wasdried, 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 for1 h at room temperature by adding 30l ofN-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlor-osilane (Pierce, Rockford, IL). After silylation, 30 l ofheptane was added. One microliter of the derivatizedsample was injected splitless by an Agilent 7683 auto-sampler (Agilent, Atlanta, GA) into an Agilent 6890 gaschromatograph following a procedure that has been des-cribed previously.31 All data were processed by Chroma-TOF (2.12) software (Leco Corp., St. Joseph, MI).Automatic peak detection and mass spectrum deconvolu-tion were performed using a peak width set to 1.5 s. Thedetected compounds were identified by comparing reten-tion indices and mass spectra with data in retention indexand mass spectra libraries.32

Acknowledgements

We thank Lise Schack at the Department of Bio-logical Chemistry at Copenhagen University forperforming assays on the UPRTase activity. We alsothank Klaus Schnackerz for his interest in thisproject. This work was supported by grants fromthe Danish and Swedish Research Councils, theSwedish Cancer Society, and the Crafoord and

Srensen Foundations.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, atdoi:10.1016/

j.jmb.2008.05.029

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a second pathway to degrade pyrimidine nucleic acid precursors in eukary

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