Saccharomyces cerevisiae Is Capable of de Novo Pantothenic Acid Biosynthesis Involving a Novel Pathway of β-Alanine Production from Spermine
2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês
10.1074/jbc.m009804200
ISSN1083-351X
AutoresW. Hunter White, Paul L. Gunyuzlu, Jeremy H. Toyn,
Tópico(s)Biotin and Related Studies
ResumoPantothenic acid and β-alanine are metabolic intermediates in coenzyme A biosynthesis. Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be rate-limiting for β-alanine and pantothenic acid biosynthesis. Overexpression ofFMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required β-alanine or pantothenic acid for growth. Furthermore, yeast genes ECM31 andYIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis. The homology of FMS1 to FAD-containing amine oxidases and its role in β-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for β-alanine biosynthesis; spe1Δ , spe2Δ ,spe3Δ , and spe4Δ are all β-alanine auxotrophs. Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the β-alanine is derived from methionine via a pathway involving spermine. These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S. cerevisiae and raise questions about these pathways in other organisms. Pantothenic acid and β-alanine are metabolic intermediates in coenzyme A biosynthesis. Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be rate-limiting for β-alanine and pantothenic acid biosynthesis. Overexpression ofFMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required β-alanine or pantothenic acid for growth. Furthermore, yeast genes ECM31 andYIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis. The homology of FMS1 to FAD-containing amine oxidases and its role in β-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for β-alanine biosynthesis; spe1Δ , spe2Δ ,spe3Δ , and spe4Δ are all β-alanine auxotrophs. Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the β-alanine is derived from methionine via a pathway involving spermine. These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S. cerevisiae and raise questions about these pathways in other organisms. Pantothenic acid (vitamin B5) is a metabolic precursor to coenzyme A (CoA) and acyl carrier protein, which are cofactors required by a large number of metabolic enzymes. Biosynthesis of pantothenic acid occurs in microbes and plants only, whereas animals obtain it in their diet. In bacteria, it is synthesized by the condensation of pantoate, derived from 2-oxoisovalerate, an intermediate in valine biosynthesis, and β-alanine, produced by the decarboxylation of l-aspartate (1Williamson J.M. Brown G.M. J. Biol. Chem. 1979; 254: 8074-8082Abstract Full Text PDF PubMed Google Scholar, 2Cronan J.E. J. Bacteriol. 1980; 141: 1291-1297Crossref PubMed Google Scholar). In Escherichia coli, four genes, panB, panC,panD, and panE, encode the four enzymes required for pantothenic acid biosynthesis, as illustrated in Fig.1 (3Jackowski S. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 687-694Google Scholar). Previous work has indicated that the yeast Saccharomyces cerevisiae requires exogenous pantothenic acid for growth (4Leonian L.H. Lilly V.G. Am. J. Bot. 1942; 29: 459-464Crossref Google Scholar). This growth requirement can be replaced by β-alanine (5Stolz J. Sauer N. J. Biol. Chem. 1999; 274: 18747-18752Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This implies that yeast have all the enzymes required for pantothenic acid biosynthesis except for aspartate-1-decarboxylase, the enzyme necessary for β-alanine biosynthesis in E. coli (1Williamson J.M. Brown G.M. J. Biol. Chem. 1979; 254: 8074-8082Abstract Full Text PDF PubMed Google Scholar, 2Cronan J.E. J. Bacteriol. 1980; 141: 1291-1297Crossref PubMed Google Scholar). Consistent with this, a structural homolog of aspartate-1-decarboxylase is absent from the proteome of yeast (6Hodges P.E. McKee A.H.Z. Davis B.P. Payne W.E. Garrels G.I. Nucleic Acids Res. 1999; 27: 69-73Crossref PubMed Scopus (192) Google Scholar), whereas structural homologs of all the other enzymes of the pantothenic acid pathway do exist in yeast. The gene ECM31, thought to be involved in cell wall maintenance (7Lussier M. White A.-M. Sheraton J. di Paolo T. Treadwell J. Southard S.B. et al.Genetics. 1997; 147: 435-450Crossref PubMed Google Scholar), has homology to panB of E. coli andAspergillus nidulans (8Kurtov D. Kinghorn J.R. Unkles S.E. Mol. Gen. Genet. 1999; 262: 115-120Crossref PubMed Scopus (17) Google Scholar). The gene YIL145c is apanC ortholog, encoding pantothenate synthase, and has been shown to be functional in E. coli (9Genschel U. Powell C.A. Abell C. Smith A.G. Biochem. J. 1999; 341: 669-678Crossref PubMed Scopus (48) Google Scholar). The putativeYHR063c gene has structural homology topanE, as noted in the Yeast Proteome Data base (6Hodges P.E. McKee A.H.Z. Davis B.P. Payne W.E. Garrels G.I. Nucleic Acids Res. 1999; 27: 69-73Crossref PubMed Scopus (192) Google Scholar). Thus, the specific absence of a gene for aspartate-1-decarboxylase may appear to be consistent with the observation, first reported almost 60 years ago, that yeast require exogenous pantothenic acid for growth (4Leonian L.H. Lilly V.G. Am. J. Bot. 1942; 29: 459-464Crossref Google Scholar). Decarboxylation of aspartate is not the only pathway for β-alanine biosynthesis. In some E. coli mutants, the source of β-alanine for pantothenic acid biosynthesis involves reduction of uracil to dihydrouracil followed by hydrolysis first to β-ureidoproprionate and second to CO2, NH3, and β-alanine (10Slotnick I.J. Weinfeld H. J. Bacteriol. 1957; 74: 122-125Crossref PubMed Google Scholar). In addition, degradation of polyamines by amine oxidases can produce β-alanine (11Hölttä E. Biochemistry. 1977; 16: 91-100Crossref PubMed Scopus (266) Google Scholar, 12Padmanabhan R. Tchen T.T. Arch. Biochem. Biophys. 1972; 150: 531-541Crossref PubMed Scopus (7) Google Scholar), effectively making β-alanine from methionine (13Large P.J. FEMS Microbiol. Rev. 1992; 8: 249-262Crossref PubMed Google Scholar). However, polyamine metabolism has never been implicated previously in pantothenic acid biosynthesis. We were therefore interested in the putative amine oxidase encoded by the yeast gene FMS1, which was originally identified as a multicopy suppressor of fen2 pantothenic acid import mutants and encodes a protein of 508 amino acids with sequence homology to FAD-containing amine oxidases (14Joets J. Pousset D. Marcireau C. Karst F. Curr. Genet. 1996; 30: 115-120Crossref PubMed Scopus (13) Google Scholar). Pantothenic acid uptake deficiency in fen2 mutants causes CoA limitation, which affects yeast growth primarily by limiting ergosterol biosynthesis, suggesting a related role for FMS1 (5Stolz J. Sauer N. J. Biol. Chem. 1999; 274: 18747-18752Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 14Joets J. Pousset D. Marcireau C. Karst F. Curr. Genet. 1996; 30: 115-120Crossref PubMed Scopus (13) Google Scholar, 15Marcireau C. Joets J. Pousset D. Guilloton M. Karst F. Yeast. 1996; 12: 531-539Crossref PubMed Scopus (12) Google Scholar). In this report we show that S. cerevisiae can synthesize β-alanine and is therefore capable of de novo biosynthesis of pantothenic acid. Furthermore, the biochemical pathway of β-alanine synthesis differs from that found in bacteria. We have found that β-alanine is formed from spermine via the amine oxidase encoded by FMS1. Thus, the β-alanine moiety of pantothenic acid is derived from methionine via S-adenosylmethionine and the polyamine pathway. These findings should facilitate the elucidation of other enzymes and metabolic intermediates involved in polyamine degradation and pantothenic acid biosynthesis and raise questions about these metabolic pathways in other organisms. The parental yeast strains BY4741 and BY4742 and their gene deletion derivatives (16Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. et al.Science. 1999; 285: 901-906Crossref PubMed Scopus (3212) Google Scholar) from theSaccharomyces Deletion Project were obtained through Research Genetics (Huntsville, AL); these strains are as follows: BY4741 (MATa his3 leu2 met15 ura3); BY4742 (MATα his3 leu2 lys2 ura3); BY4741–0595 and BY4742–10595 (fms1Δ); BY4741–5757 and BY4742–15757 (fen2Δ); BY4741–3316 and BY4742–13316 (ecm31Δ); BY4741–2304 and BY4742–12304 (YIL145cΔ); BY4741–5034 and BY4742–15034 (spe1Δ); BY4741–1743 and BY4742–11743 (spe2Δ); BY4741–5488 and BY4742–15488 (spe3Δ); BY4741–6945 and BY4742–16945 (spe4Δ). JHT14 (MATα abz1Δ::HIS3 his3 trp1 ura3) was made during this study. The abz1Δ::HIS3allele was constructed by micro-homologous recombination (17Manivasakam P. Weber S.C. McElver J. Schiestl R.H. Nucleic Acids Res. 1995; 23: 2799-2800Crossref PubMed Scopus (151) Google Scholar) with a DNA construct made in a single step by the polymerase chain reaction, using oligonucleotide polymerase chain reaction primers (Sigma-Genosys, The Woodlands, Texas) designed as previously described for the trpΔ mutants (18Toyn J.H. Gunyuzlu P.L. White W.H. Thompson L.A. Hollis G.F. Yeast. 2000; 16: 553-560Crossref PubMed Scopus (93) Google Scholar). Growth and manipulation of yeast strains (19Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics: Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar, 20Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar) was on "YNB-P" medium, which was mixed from the individual chemicals. YNB-P had the same recipe as "Yeast Nitrogen Base with ammonium sulfate" (Difco, Detroit, MI), except that pantothenic acid was omitted. Amino acid supplements (18Toyn J.H. Gunyuzlu P.L. White W.H. Thompson L.A. Hollis G.F. Yeast. 2000; 16: 553-560Crossref PubMed Scopus (93) Google Scholar) and 2% glucose were also present in YNB-P medium. Pantothenic acid and other supplements were added to YNB-P as indicated in "Results" and the figure legends. YPD medium was 2% glucose, 2% bactopeptone, and 1% yeast extract (Difco). Pantothenic acid, β-alanine, spermine, spermidine, and putrescine (Sigma) were prepared as stock solutions in water and used in media at the concentrations indicated in the figure legends. Yeast strain JHT14 was transformed with a yeast high copy library (21Carlson M. Botstein D. Cell. 1982; 28: 145-154Abstract Full Text PDF PubMed Scopus (926) Google Scholar) and ∼5 × 104 Ura+ transformants were pooled, divided into aliquots, and stored in 25% glycerol at −70 °C. Transformants were then spread at a density of 105 Ura+ cells per 10-cm Petri dish on synthetic agar medium containing 2% glucose, but lacking uracil, adenine, histidine, methionine, and pantothenic acid. After incubation for 3 days at 30 °C, rapidly growing colonies occurred at a frequency of ∼1.5 × 10−4. The rapid growth phenotype of 37 of 55 colonies tested was found to be plasmid-dependent, based on the lack of growth on a selective medium containing 5-fluoroorotic acid and uracil (22Boeke J.D. LaCroute F. Fink G.R. Mol. Gen. Genet. 1984; 197: 345-346Crossref PubMed Scopus (1712) Google Scholar). Ten of these plasmids were recovered from yeast by transformation of E. coli and confirmed to confer the rapid growth phenotype on selective medium when reintroduced into yeast strain JHT14. Based on comparison with sequence data in theSaccharomyces Genome Data base, six plasmids contained theABZ1 locus, and four plasmids contained the FMS1locus. The ABZ1 plasmids enhanced growth only because of the low concentration of paraaminobenzoic acid used in the medium, and they were not studied further. E. coli strains DH5α and DH10B (Life Technologies, Inc.) were used for DNA manipulations by standard methods (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmids were introduced into yeast using lithium acetate (24Gietz R.D. Schiestl R.H. Methods Mol. Cell. Biol. 1995; 5: 255-269Google Scholar). Yeast DNA isolation for recovery of plasmids in E. coli was carried out using the Yeast Plasmid Isolation kit (Bio 101, Carlsbad, CA). The FMS1 coding sequence was amplified by polymerase chain reaction using oligonucleotide primers Fms1For-Xho (5′-ccctcgagatgaatacagtttcaccag-3′) and Fms1Rev-Bam (5′-ttggatccctatttcagtaagtcag-3′) and ligated into theSalI and BamHI sites of YEp195AC (25Gunyuzlu P.L. White W.H. Davis G.L. Hollis G.F. Toyn J.H. Mol. Biotechnol. 2000; 15: 29-37Crossref PubMed Scopus (10) Google Scholar) to create the ADH1-FMS1 overexpression vector. The E39Q substitution was made using QuikChange (Stratagene, La Jolla, CA) and mutagenic primers FmsE39Qupper (5′-gtcttgttcttcaggccagagatc-3′) and FmsE39Qlower (5′-gatctctggcctgaagaacaagac-3′). The DNA sequence of the entire mutant open reading frame was confirmed subsequently. Log phase cultures of strain BY4742–10595 (fms1Δ) or strain BY4742–13316 (ecm31Δ) containing vector YEp195AC were prepared in synthetic medium lacking uracil and washed by centrifugation in water, and ∼105 cells were spread on 10-cm Petri dishes containing synthetic agar medium lacking uracil and pantothenic acid. Log phase cultures of strain BY4742 and fen2Δ, fms1Δ, and ecm31Δ deletion derivatives harboring either YEp195AC or the ADH1-FMS1overexpression plasmid were prepared in synthetic medium lacking uracil, washed by centrifugation in water, and spotted onto theecm31Δ and fms1Δ "lawns" at a density of ∼107 cells per 5 μl. Plates were incubated at 30 °C for 2 days, after which time "halos" of growing lawn cells formed around spots of cells that excreted pantothenic acid or downstream metabolites into the medium. A high copy yeast genomic DNA library was screened for genes that enhanced growth in the absence of pantothenic acid, and plasmids containing genomic DNA in the region of the FMS1 locus were found. To determine whether FMS1, rather than other DNA sequences in these plasmids, was responsible for the enhanced growth, the FMS1 open reading frame was subcloned into an expression vector under the control of the ADH1 promoter and confirmed to have a DNA sequence identical to the published sequence (Ref. 14Joets J. Pousset D. Marcireau C. Karst F. Curr. Genet. 1996; 30: 115-120Crossref PubMed Scopus (13) Google Scholar, GenBankTM accession number X81848). This plasmid was introduced into yeast, and it was found that ADH1-FMS1, but not the empty vector, could enhance the growth of yeast on medium lacking pantothenic acid (Fig. 2). As shown above, yeast grew well in the absence of pantothenic acid when FMS1 was overexpressed. This finding prompted us to test the currently available deletion strains, fms1Δ, ecm31Δ,YIL145cΔ, and fen2Δ for pantothenic acid and β-alanine auxotrophy (see Fig. 1). The deletion strains and parental strain BY4742 were plated on medium lacking pantothenic acid and β-alanine or on medium supplemented with these compounds (Fig.3 A). The fms1Δstrain required either pantothenic acid or β-alanine for growth. This is consistent with the results for overexpression of FMS1; overexpression of FMS1 enhanced growth in the absence of pantothenic acid/β-alanine, whereas the fms1Δ deletion totally blocked growth in the absence of pantothenic acid/β-alanine. In the same experiment, the ecm31Δ andYIL145cΔ strains could utilize pantothenic acid but differed from the fms1Δ strain because they could not grow on β-alanine. Neither the fen2Δ nor the parental strain required these supplements. Other potential metabolites, including β-ureidoproprionate, 5,6-dihydrouracil, l-aspartic acid, and 1,3-diaminopropane (100 μm) did not support growth of the deletion strains (data not shown). The same results were also obtained using a different parental strain, BY4741, and its deletion derivatives (data not shown). Thus, based on the auxotrophic phenotypes,FMS1 is required for β-alanine production, whereasECM31 and YIL145c are required downstream in the pantothenic acid pathway (Fig. 1). Further evidence that FMS1 functions in the same pathway asECM31 was obtained from a complementation analysis using theADH1-FMS1 overexpression plasmid. This plasmid was introduced into fms1Δ, ecm31Δ, andfen2Δ strains, which were then tested for growth in the absence of β-alanine (Fig. 3 B). Growth occurred in thefms1Δ and fen2Δ strains, but not theecm31Δ strain, indicating that FMS1 is dependent on ECM31 for pantothenic acid biosynthesis. Thus,S. cerevisiae does not require exogenous pantothenic acid or β-alanine in the medium for growth, and FMS1 activity is rate-limiting for β-alanine biosynthesis under the conditions used. Dramatically increased metabolic activity in the pantothenic acid pathway caused by FMS1 overexpression can be detected in cross-feeding experiments, in which cells excreting excess pantothenic acid cause halos of growth in lawns of pantothenic acid auxotrophs. Dense spots of wild-type,fen2Δ, fms1Δ, and ecm31Δ strains containing either the ADH1-FMS1 overexpression vector or the empty vector were placed on lawns of either ecm31Δ orfms1Δ cells on medium lacking pantothenic acid (Fig.4). After incubation, halos formed around each of the FMS1-overexpressing strains, with the exception of the ecm31Δ strain, on both lawns. No halos formed around strains harboring empty vector. The simplest explanation for the halos is that FMS1 overexpression results in excretion of excess pantothenic acid, which is then taken up by the lawn cells, allowing them to grow. Overexpression of FMS1 in theecm31Δ strain did not result in a halo, further confirming that FMS1 and ECM31 function in the same pathway. Although it is required for β-alanine production,FMS1 encodes a protein that has no structural homology to bacterial aspartate-1-decarboxylases. Instead, Fms1p has homology to FAD-containing amine oxidases (14Joets J. Pousset D. Marcireau C. Karst F. Curr. Genet. 1996; 30: 115-120Crossref PubMed Scopus (13) Google Scholar) and likewise contains a GXGXXG dinucleotide-binding motif similar, for example, to Candida albicans Cbp1p, human monoamine oxidases A and B, and the peroxisomal acetylspermidine oxidase Aso1p of Candida boidinii (Fig.5 A). To assess the role of FAD in β-alanine production, we made the E39Q substitution mutant, equivalent to the substitution that was shown to abolish FAD binding and catalytic activity of monoamine oxidase B (26Kwan S.-W. Lewis D.A. Zhou B.P. Abell C.W. Arch. Biochem. Biophys. 1995; 316: 385-391Crossref PubMed Scopus (29) Google Scholar, 27Zhou B.P. Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. 1995; 270: 23653-23660Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 28Zhou B.P. Wo B. Kwan S.-W. Abell C.W. J. Biol. Chem. 1998; 273: 14862-14868Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The resultingADH1-fms1(E39Q) expression plasmid was introduced into thefms1Δ strain, and transformants were tested for growth in the absence of β-alanine and pantothenic acid (Fig. 5 B). The E39Q mutant did not complement the β-alanine and pantothenic acid auxotrophy of the fms1Δ strain, consistent with a role for FAD in the mechanism of β-alanine production. The sequence homology of Fms1p to amine oxidases and the apparent role of FAD in β-alanine production by Fms1p suggested that polyamines could provide the substrates for Fms1p. We therefore tested deletion mutants of the polyamine pathway (29Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3236) Google Scholar, 30Glansdorff N. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 408-433Google Scholar) for β-alanine auxotrophy. Parental, spe1Δ, spe2Δ,spe3Δ, spe4Δ, fms1Δ, andecm31Δ strains were plated on medium lacking pantothenic acid, β-alanine, and polyamines or on medium supplemented with pantothenic acid, β-alanine, spermine, spermidine, or putrescine (Fig. 6). All four of thespeΔ mutants were able to grow when one of the compounds spermine, β-alanine, or pantothenic acid was added to the medium. In addition, spe1Δ and spe3Δ could also grow on spermidine, and spe1Δ could grow on putrescine. As expected, the fms1Δ and ecm31Δ strains could grow on pantothenic acid but could not utilize any of the polyamine compounds. Thus, biosynthesis of β-alanine and pantothenic acid is dependent on the polyamine biosynthetic pathway, consistent with production of β-alanine via polyamine degradation (11Hölttä E. Biochemistry. 1977; 16: 91-100Crossref PubMed Scopus (266) Google Scholar, 12Padmanabhan R. Tchen T.T. Arch. Biochem. Biophys. 1972; 150: 531-541Crossref PubMed Scopus (7) Google Scholar, 13Large P.J. FEMS Microbiol. Rev. 1992; 8: 249-262Crossref PubMed Google Scholar). The source of the carbon atoms in β-alanine would therefore be from methionine via spermine. Based on these results, the relationship of the polyamine pathway to pantothenic acid biosynthesis and the key genes involved are illustrated in Fig. 7. The speΔ strains were not able to grow on the potential polyamine degradation metabolite 1,3-diaminopropane (data not shown), apparently ruling out this compound as an intermediate in β-alanine biosynthesis.Figure 7The pantothenic acid pathway in yeast. A, the diagram shows the relationship of yeast genes to pantothenic acid biosynthesis, inferred from the experiment in Fig. 6. The dashed arrow between spermine and β-alanine represents the novel connection between polyamine degradation and pantothenic acid biosynthesis in yeast, involving FMS1 and additional unidentified genes. B, chemical structures for methionine, decarboxyadenosylmethionine (dcAdoMet), putrescine, spermidine, spermine, 3-aminopropanal, β-alanine, and (R)-pantothenate. The bold lines represent the three-carbon moiety that is derived from methionine in yeast.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Yeast have been reported to require a supplement of either pantothenic acid or β-alanine, from which it has been inferred that they cannot synthesize pantothenic acid de novo (4Leonian L.H. Lilly V.G. Am. J. Bot. 1942; 29: 459-464Crossref Google Scholar, 5Stolz J. Sauer N. J. Biol. Chem. 1999; 274: 18747-18752Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Contrary to this, we found that overexpression of the yeast geneFMS1, encoding a putative amine oxidase, allowed strong growth on medium lacking pantothenic acid. Furthermore, whenFMS1 was overexpressed under the control of theADH1 promoter, excess pantothenic acid was excreted from the cells. Thus, yeast clearly have the capacity to synthesize pantothenic acid de novo when FMS1 is overexpressed. To eliminate the possibility that the pantothenic acid biosynthesis was a metabolic abnormality caused by FMS1 overexpression, we analyzed gene deletion mutants. The fms1Δ strains were auxotrophic for β-alanine and could grow when supplemented with either β-alanine or pantothenic acid. Deletions in genes that have structural homology to the bacterial genes of the pantothenic acid pathway, ECM31 and YIL145c (see Fig. 1), caused pantothenic acid auxotrophy, but these strains did not grow on a β-alanine supplement, indicating that these genes are downstream in the pathway (see Fig. 1). These auxotrophic phenotypes indicate thatFMS1, ECM31, and YIL145c are normally involved in pantothenic acid biosynthesis. Direct evidence thatFMS1 and ECM31 are in the same pathway came from the finding that FMS1 requires ECM31 activity to make pantothenic acid; the ecm31Δ deletion eliminated both the ability of FMS1 to enhance growth in the absence of pantothenic acid and also eliminated its ability to cause pantothenic acid excretion. Thus, pantothenic acid biosynthesis is a natural part of metabolism in S. cerevisiae, and production of the β-alanine required involves a putative amine oxidase, encoded byFMS1. Three different enzymatic pathways have been shown to produce β-alanine: decarboxylation of aspartate (1Williamson J.M. Brown G.M. J. Biol. Chem. 1979; 254: 8074-8082Abstract Full Text PDF PubMed Google Scholar, 2Cronan J.E. J. Bacteriol. 1980; 141: 1291-1297Crossref PubMed Google Scholar), degradation of pyrimidines (10Slotnick I.J. Weinfeld H. J. Bacteriol. 1957; 74: 122-125Crossref PubMed Google Scholar), and degradation of polyamines (13Large P.J. FEMS Microbiol. Rev. 1992; 8: 249-262Crossref PubMed Google Scholar). The polyamine pathway has not been implicated previously in pantothenic acid biosynthesis, and spermine, in particular, has no previously identified physiological function in yeast (31Hamasaki-Katagiri N. Katagiri Y. Tabor C.W. Tabor H. Gene. 1998; 210: 195-201Crossref PubMed Scopus (59) Google Scholar). The involvement of the polyamine pathway is suggested by the Fms1p amino acid sequence, which has structural homology to FAD-containing amine oxidases (14Joets J. Pousset D. Marcireau C. Karst F. Curr. Genet. 1996; 30: 115-120Crossref PubMed Scopus (13) Google Scholar), some of which are involved in the oxidative degradation of polyamines (4Leonian L.H. Lilly V.G. Am. J. Bot. 1942; 29: 459-464Crossref Google Scholar). In addition, we showed that a FAD binding site mutant of FMS1, E39Q, did not complement the fms1Δ mutant, consistent with a role for oxidation by the Fms1p protein. We therefore investigated deletion mutants of the polyamine pathway to see whether this pathway is required for β-alanine synthesis. Indeed, spe1Δ,spe2Δ, spe3Δ, and spe4Δ mutants were all auxotrophic for β-alanine on a medium that lacked polyamines. This showed that synthesis of spermine is required for β-alanine biosynthesis in yeast. A more detailed analysis of which polyamine pathway intermediates could support growth of thespeΔ and fms1Δ mutants confirmed this conclusion (Fig. 6). Thus, in yeast, β-alanine is derived from methionine via spermine, making polyamine degradation part of pantothenic acid biosynthesis (Fig. 7). We found that the auxotrophic phenotypes of the speΔmutants for polyamines were readily observable on minimal synthetic medium in the absence of β-alanine or pantothenic acid (Fig.6). This contrasts with previous reports, in which special precautions in medium preparation were required, such as HCl washing of glassware and avoidance of autoclave use, to eliminate contaminating amines, and in which many cell divisions were required to deplete intracellular pools of polyamines (32Balasundaram D. Tabor C.W. Tabor H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5872-5876Crossref PubMed Scopus (97) Google Scholar). The difference is simply in the presence or absence of β-alanine or pantothenic acid; in their absence, a relatively high level of polyamine metabolism is required to meet β-alanine requirements, such that contaminated glassware and intracellular pools do not make a significant contribution. In the presence of β-alanine or pantothenic acid, as customary in yeast media, low levels of contaminating polyamines are sufficient for essential processes, such as hypusine synthesis (33Park M.H. Joe Y.A. Kang K.R. J. Biol. Chem. 1998; 273: 1677-1683Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), which are unrelated to the pantothenic acid pathway. Amine oxidases have been shown to catalyze a number of different degradation reactions for polyamines, producing various aldehydes and amines, such as 3-aminopropanal and 1,3-diaminopropane, respectively (13Large P.J. FEMS Microbiol. Rev. 1992; 8: 249-262Crossref PubMed Google Scholar). Thus, the simplest hypothesis is that the Fms1p enzyme converts spermine to 3-aminopropanal and spermidine and that aldehyde dehydrogenases, for which there are seven genes in yeast (34Wang X. Mann C.J. Bai Y. Ni L. Weiner H. J. Bacteriol. 1998; 180: 822-830Crossref PubMed Google Scholar), would be required to convert the 3-aminopropanal to β-alanine. A less direct route between spermine and β-alanine could, in principle, involve the intermediate 1,3-diaminopropane (13Large P.J. FEMS Microbiol. Rev. 1992; 8: 249-262Crossref PubMed Google Scholar). However, this compound was not able to support growth of the speΔ mutants in the absence of β-alanine and therefore appears not to be on the pathway in yeast. The simple phenotype of β-alanine auxotrophy in yeast will help identify the metabolic intermediates and additional enzymes involved. It may seem unexpected that yeast would have the capacity to make pantothenic acid and yet require a supplement for efficient growth on customary yeast media. In fact, on medium containing glycerol or acetate as the sole carbon source, we found that pantothenic acid and β-alanine were not rate-limiting for growth (data not shown). Thus, FMS1activity is growth-limiting only on glucose medium. This simple observation may explain the carbon source-dependent phenotype (catabolite repression) reported for the fen2pantothenate transporter mutant (15Marcireau C. Joets J. Pousset D. Guilloton M. Karst F. Yeast. 1996; 12: 531-539Crossref PubMed Scopus (12) Google Scholar). In the light of the finding that pantothenic acid biosynthesis is a natural part of yeast metabolism, we propose that growth of the fen2 mutant, which cannot absorb pantothenic acid from the medium, depends on pantothenic acid synthesis inside the yeast cells. The fen2 mutant would therefore be growth-limited on glucose because of insufficient FMS1expression caused by the presence of glucose. Likewise, wild-type strains would depend on internal synthesis when pantothenic acid was absent from the medium and would be growth-limited by insufficientFMS1 expression on glucose medium. These observations suggest that FMS1 activity is regulated and raise questions concerning the mechanism of regulation of the pantothenic acid pathway in yeast. The finding that β-alanine biosynthesis is different between yeast and bacteria raises questions as to how other organisms, such as fungi and plants, make β-alanine. At the present time in the public sequence data bases there are over a dozen identifiable aspartate-1-decarboxylase genes from different prokaryotic species, whereas this enzyme does not appear to be present in eukaryotic species. In contrast, proteins of significant sequence homology to Fms1p can be found in eukaryotes, in particular in plants, but not in prokaryotes. The closest sequence similarity to Fms1p is in Cbp1p from the yeast C. albicans, a protein with steroid binding activity (14Joets J. Pousset D. Marcireau C. Karst F. Curr. Genet. 1996; 30: 115-120Crossref PubMed Scopus (13) Google Scholar, 35Malloy P.J. Zhao X. Madani M.D. Feldman D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1902-1906Crossref PubMed Scopus (56) Google Scholar). This suggests that plants and lower eukaryotes generally produce β-alanine and hence CoA by a polyamine degradation pathway, as described here for yeast. We thank our colleagues at DuPont Pharmaceuticals: Greg Hollis, Karyn O'Neil, and Shaoxian Sun for their critical evaluation and help in writing the manuscript and Julie Bunville, Karen Krakowski, and Laura Bolling for DNA sequencing.
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