Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK1
Author for correspondence: Peter A. Meacock. Tel: +44 116 252 2261. Fax: +44 116 252 5101. e-mail: mea{at}le.ac.uk
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ABSTRACT |
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Keywords: acetolactate synthase, thiamin pyrophosphate, THI1, ILV2, S. cerevisiae
Abbreviations: AHAS, acetohydroxyacid synthase; ALS, acetolactate synthase; PDC, pyruvate decarboxylase; POX, pyruvate oxidase; TK, transketolase; TPP, thiamin pyrophosphate
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INTRODUCTION |
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Thiamin is synthesized in micro-organisms and plants from two precursors, hydroxyethyl thiazole phosphate (HET-P) and hydroxymethyl pyrimidine pyrophosphate (HMP-PP), that are condensed together to form thiamin monophosphate (TMP). In the bakers yeast Saccharomyces cerevisiae TMP is then dephosphorylated to free thiamin, which is subsequently pyrophosphorylated to form TPP, the endproduct of the pathway. Although some of the enzymes and genes involved in thiamin biosynthesis have been characterized in yeast (reviewed by Begley, 1996 ; Estramareix & David, 1996
; Hohmann & Meacock, 1998
), much is still unknown. Therefore, in order to learn more about thiamin biosynthesis and metabolism in S. cerevisiae we have embarked upon a genetic analysis of strains carrying thiamin auxotrophic mutations. These included strain Y02587, which carries the first thiamin auxotrophic mutation isolated in S. cerevisiae, thi1 (Mortimer & Hawthorne, 1966
). Apart from its nutritional requirements and localization of the thi1 mutation to the right arm of chromosome XIII, no other information was available regarding this strain.
In this paper we describe the isolation of the wild-type THI1 gene. Surprisingly, we found that THI1 is allelic with ILV2, which encodes AHAS. Here we show that thiamin auxotrophy can arise from an alteration in cofactor dependence of a TPP-utilizing enzyme rather than through mutation of a structural or regulatory gene of the biosynthetic pathway. We also report that an amino acid residue that does not form part of the consensus TPP-binding motif has a role in determining the cofactor dependency of the enzyme AHAS.
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METHODS |
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Plasmids used or constructed in this study are listed in Table 1. To construct the ilv2
::URA3 deletion allele, DNA of plasmid pKB19c carrying the minimal thi1-complementing fragment was digested with the restriction endonuclease XbaI and religated to itself, thereby deleting 1·352 kb of the gene and creating the plasmid pKB19h (Fig. 1
). A DNA fragment carrying the URA3 gene was released from pKB22a by XbaI digestion and ligated into XbaI-cut pKB19h to give plasmid pKB22, carrying the ilv2::URA3 construct. This plasmid was then digested with HindIII and EcoRI, to recover the 2·29 kb disruption cassette for transformation into yeast.
Yeast procedures.
All standard media and genetic procedures were as described by Sherman (1991) . For selective growth, yeast was cultured in SD medium supplemented with the required amino acids and bases. Sporulation was carried out by growth on acetate-based medium, following transfer from rich pre-sporulation medium. To test for thiamin auxotrophy, strains were grown in Wickerhams minimal medium (Wickerham, 1951
), with or without thiamin, and supplemented with appropriate amino acids and bases. High-efficiency transformations of yeast were performed by the lithium acetate procedure as described by Geitz et al. (1995)
. For disruption of the ILV2 locus the one-step gene disruption method was used (Rothstein, 1983
). Gap repair was used as described by Orr-Weaver & Szostak (1983)
to recover the ilv2-thi1 allele.
DNA manipulations.
Standard DNA handling procedures were carried out according to protocols described in Sambrook et al. (1989) . Total yeast chromosomal DNA of high quality, for use in Southern blot analysis, was prepared from spheroplasts (Cryer et al., 1975
). DNA samples were sequenced on an ABI model 373A DNA sequencer, following sample preparation using the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit. The primer for sequencing DNA cloned into the BamHI site in YCp50 DNA was obtained from New England Biolabs; 1223, pBR322 BamHI site primer (clockwise). Custom-made primers were supplied by PNACL at Leicester University and are listed in Table 2
.
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Acetohydroxyacid synthase (AHAS) assay.
Yeast strains were grown overnight (25 or 50 ml) to a concentration of 13x107 cells ml-1, in Wickerhams minimal medium supplemented with tryptophan, histidine, adenine, isoleucine and valine. Cells were harvested at 5000 r.p.m. for 5 min, washed twice in sterile distilled H2O and resuspended in 1·0 ml 0·1 mM Tris/HCl buffer (pH 7·5), 0·1 M NaCl, 0·1 mM EDTA at 4 °C containing the protease inhibitors chymostatin (1 µg ml-1), PMSF (1 mM) and pepstatin (1 µM). These were permeabilized by adding 100 µl chloroform and vortexing for 30 s, collected by centrifugation and the supernatant was decanted. After resuspension in 1·0 ml of the same buffer the total protein concentration was measured using the Bio-Rad Bradford Reagent method. AHAS was assayed via its acetolactate synthase (ALS) activity. Acetolactate production was measured at 30 °C for 30 min, using a method based on that of Ryan & Kohlhaw (1974) . For each assay two tubes were set up, A and B, each containing a mixture of 40 mM sodium pyruvate, 10 mM MgCl2, 100 mM Tris/HCl (pH 7·5) and varying concentrations of TPP (0200 µM), to a total volume of 100 µl. The reactions were started by the addition of
0·5 mg permeabilized cell protein to each tube. They were stopped by the addition of 50 µl 3 M sulphuric acid to tube A and 150 µl 6 M sodium hydroxide to tube B. The yield of acetolactate produced was determined by converting it to acetoin. This involved measuring the total amount of acetoin produced (tube A) and subtracting the amount of background acetoin produced by the cells (tube B). Acetolactate was converted into acetoin by a decarboxylation step that involved heating the sample in an acid environment. Therefore tube A was incubated at 60 °C for 30 min, after which the sample was made basic by the addition of 100 µl 12 M sodium hydroxide. Acetoin was measured according to the method of Westerfeld (1945)
. Each sample was made up to a total volume of 1 ml with sterile distilled water, then 200 µl 0·5% creatine was added followed by 200 µl 5%
-naphthol (freshly made up in 2·5 M sodium hydroxide) and the solution mixed by inverting the tubes several times. The colour was allowed to develop for 1 h at room temperature, the samples were centrifuged for 2 min and then their absorbance at 540 nm was measured against a blank made up of 1 ml water, 200 µl creatine and 200 µl
-naphthol. The ALS activity was calculated as acetoin produced per mg of permeabilized cell protein per minute.
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RESULTS |
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In order to test whether the thi1 mutation was allelic with other known mutants which gave thiamin auxotrophy, thi2 (Kawasaki et al., 1990 ), thi3 (Nishimura et al., 1992
), thi4 (Praekelt et al., 1994
) and thi6 (Nosaka et al., 1994
), complementation analysis was performed. Appropriate crosses were made between the thi1 mutant and each of the other mutant strains, and the heterozygous diploids were tested for thiamin requirement; all of the diploids were prototrophic for thiamin. Thus thi1 is not allelic with the thiamin mutations thi2, thi3, thi4 or thi6.
DNA clones that complemented the thi1 mutation were obtained on the basis of their ability to rescue growth of the mutant on thiamin-deficient medium. A thi1 ura3 strain (KBY6) was transformed to uracil prototrophy using a YCp50-based S. cerevisiae genomic DNA library (Rose et al., 1987 ). Among the several thousand Ura+ transformants recovered, 19 were thiamin prototrophs; in all cases Ura- segregants that had lost the transforming clones displayed thiamin auxotrophy, indicating that the prototrophic phenotype of the transformants was caused by the introduced library plasmid and not by reversion of the original thi1 mutation. Plasmid DNAs were recovered into E. coli from two of the transformants and these were found to complement the thi1 mutation when retransformed back into the KBY6 strain, confirming that each carried the THI1 gene.
Restriction enzyme digestions revealed that the yeast genomic DNA fragments in the two plasmids were identical. Subcloning of the insert DNA from one plasmid (pKB19) gave a 5·185 kb HindIIIXhoI fragment that was able to complement the thi1 mutation when reintroduced into KBY6 on vector YCp50 (pKB19c, Fig. 1). Nucleotide sequence data were obtained from the ends of the insert DNA and compared with the EMBL nucleotide database. The sequence of the insert DNA was found to match the region of yeast chromosome XIII containing the ORF YMR108w, previously characterized as the ILV2 gene. Analysis of the sequence of the 5·185 kb DNA fragment revealed three other potential ORFs, all smaller than 420 bp in length (Fig. 1
). Further dissection of the DNA fragment carried in pKB19c produced the smaller derivatives pKB19dh (Fig. 1
); phenotypic analysis showed that none of these was able to rescue the thiamin auxotrophy of thi1. These subclones included pKB19g, carrying only the internal 2·0 kb EcoRI fragment, and the reciprocal derivative, pKB19f, that had this fragment deleted. These data suggested that the ORF responsible for thi1 complementation spanned one or both of these EcoRI sites and thus ruled out ORFs 1 and 2 as candidates for the THI1 gene, as both were contained entirely within the 2·0 kb fragment. Also ORF 3 (YMR107w) was eliminated because it was located entirely outside this region. Therefore the only ORF which spanned one or both of the EcoRI sites was YMR108w, the ILV2 gene.
Allelism of thi1 and ILV2
Normally mutations at the ILV2 locus cause auxotrophy for the branched-chain amino acids isoleucine and valine (Falco & Dumas, 1985 ). However, the thi1 strain Y02587 did not require isoleucine and valine for growth (Fig. 2
). Furthermore, a strain (STX573-3C) previously confirmed to carry a bona fide ilv2 mutation showed isoleucine and valine auxotrophy but no requirement for thiamin; i.e. the same phenotype as strain W303
ilv2 (Fig. 2
). We also found that the thi1-complementing clone, pKB19c, conferred isoleucine and valine prototrophy when transformed into strain STX573-3C, indicating that it carried an ILV2 allele. Likewise a bona fide ILV2 clone, pE16/8-C4 (Polaina, 1984
), was able to restore thiamin prototrophy when transformed into the thi1 strain KBY6. This gave an intriguing paradox: the thi1 mutant was not isoleucine and valine requiring and the ilv2 mutant was not thiamin requiring, yet both mutations were complemented to their respective prototrophies by the same fragment of DNA. One explanation could be that YMR108w was the wild-type locus for one of the mutations and a suppressor of the other. However the complementing fragments were carried on low-copy-number vectors, which made suppression unlikely. It was therefore important to delete this locus and examine the phenotype of the resultant ilv2
null mutant. Additionally, examination of the meiotic segregants of diploids made by crossing strains carrying the ilv2
and thi1 mutations would help to resolve whether they were allelic.
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Because of this observation we decided to reinvestigate the phenotype of strains carrying the original thi1 mutation. We found that the haploid thi1 strain did not in fact require thiamin for growth when isoleucine and valine were supplied in the medium (Fig. 2). Therefore the true phenotype of the thi1 mutation is that of thiamin auxotrophy only in the absence of isoleucine and valine.
Finally, we crossed the strain W303ilv2 carrying the ilv2
::URA3 allele and the thi1 strain KBY6. The heterozygous diploid exhibited the thi1 phenotype (thiamin auxotrophy in the absence of isoleucine and valine). Sporulation and analysis of 10 meiotic tetrads was carried out (Table 3
). In the presence of isoleucine and valine 4:0 segregation of Thi+:Thi- phenotypes was observed for all tetrads. However, in the absence of isoleucine and valine only two spores from each tetrad were viable with thiamin supplementation, and no spores were viable in the absence of thiamin as well as isoleucine and valine. Taken together these observations implied that the ilv2
and thi1 mutations showed 2:2 segregation consistent with their being allelic. Moreover the absence of any non-parental ditype or tetratype tetrads confirmed that the thi1 and ilv2
mutations were tightly linked.
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To test this hypothesis we decided to investigate the molecular nature of the thi1 mutation and to characterize the AHAS enzyme produced from the ilv2-thi1 allele. Recovery of the mutant allele was achieved using the gap repair technique (Orr-Weaver & Szostak, 1983 ). Cells of the thi1 ura3 strain KBY6 were transformed to uracil prototrophy with XbaI-linearized DNA of the ARS-CEN-based plasmid pKB19h (Fig. 1
) which had part of the ILV2 sequence deleted from the insert DNA. The in vitro-generated gap in the transforming DNA was repaired by in vivo recombination with the homologous chromosomal sequences. Plasmid DNAs from two different Ura+ transformants were recovered into E. coli and then transformed back into the ura3 ilv2
strain KBY8. Transformants carrying each plasmid clone gave the thi1 phenotype, thiamin auxotrophy in the absence of isoleucine and valine, confirming that we had recovered the ilv2-thi1 allele.
In order to establish exactly where the thi1 mutation was located and to determine its effect on the AHAS amino acid sequence, we carried out nucleotide sequencing of the mutant ORF of one plasmid, pKB24, using oligonucleotide primers (Table 2). Only a single nucleotide difference was found within the 2064 bp sequence of the ilv2-thi1 allele when compared to the published ILV2 sequence (Falco et al., 1985
). The mutation at nucleotide position 528 of the ILV2 ORF (YMR108w) was a T to G transversion changing codon 176 from GAT to GAG, and thereby replacing the normal aspartate residue with the related glutamate, i.e. D176E. Moreover, the mutation also created a new cleavage site for the restriction endonuclease StuI: 5'-ATGCCT-3' to 5'-AGGCCT-3'. However the thi1 mutation was located 100 bp upstream from the 5' XbaI site used for the gap repair cloning of the ilv2-thi1 allele. Thus recombination between the transforming gapped plasmid and ilv2-thi1 sequences must have extended well beyond the ends of the linearized plasmid DNA.
To confirm that strains carrying the original ilv2-thi1 allele did contain this particular mutation within the ILV2 ORF we carried out a Southern blot analysis to look for the presence of the additional StuI site. Genomic DNAs were isolated from the thi1 strain KBY6 and two Thi+ Ilv2+ strains, DG622 and W303B; these were digested with restriction enzyme StuI and subjected to Southern blot analysis using part of the ILV2 gene as a hybridization probe (Fig. 3). Inspection of the published sequence of chromosome XIII (Bowman et al., 1997
) showed that ILV2 is contained within a 23·279 kb StuI fragment. The DNAs of strains W303B and DG622 both yielded a single hybridizing fragment of approximately 23 kb (Fig. 3
, lanes 1 and 3). However, DNA of strain KBY6 yielded two smaller hybridizing fragments, of approximately 5 kb and 19 kb (Fig. 3
, lane 2). These corresponded to the sizes of fragments (4·563 kb and 18·716 kb) that would be produced if an extra StuI restriction site had been created at nucleotide 528 of the ILV2 ORF. Therefore the mutation generating the StuI restriction site was present in strains carrying the original thi1 allele.
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AHAS activity in thi1 mutants
We have postulated that the thi1 mutant might produce a variant of AHAS that has altered dependence for its cofactor TPP, so that the enzyme can only function in branched-chain amino acid biosynthesis when intracellular TPP levels are raised by exogenous supply of thiamin. To test this hypothesis we assayed AHAS activity, and its TPP dependency, in yeast strains carrying various alleles of ILV2. Acetolactate synthase (ALS) activity, one of the two activities of AHAS, was measured in the KBY8 strain carrying different ILV2 alleles on low-copy-number plasmids: pKB19c (ILV2), pKB24 (ilv2-thi1), pKB30 (ilv2-thi1:sdm) and YCp50 (ilv2).
Permeabilized cells carrying the wild-type ILV2 allele (pKB19c) were found to have high levels of activity that showed little dependence on TPP concentration over the range 50200 µM (Fig. 4). Even with no additional TPP high levels of acetolactate were produced, indicating that the cells contained sufficient endogenous TPP for almost full ALS activity. In contrast, permeabilized cells carrying the ilv2-thi1 allele (pKB24) showed no or little ALS activity in the absence of exogenous TPP; addition of the cofactor stimulated activity but never to the very high levels recorded in the ILV2 cells. The data obtained from the enzyme produced by the site-directed mutagenesis clone pKB30, ilv2-thi1:sdm, were virtually identical to those obtained for AHAS produced from the original ilv2-thi1 mutant allele. Again, there was no measurable ALS activity when no exogenous TPP was added to the assay mixture, but activity was detected as the TPP concentration was increased.
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DISCUSSION |
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The structures of three enzymes that use TPP as a cofactor have been resolved by X-ray crystallography. These are PDC from S. cerevisiae (Dyda et al., 1993 ), TK from S. cerevisiae (Lindqvist et al., 1992
; Nilsson et al., 1993
) and POX from Lactobacillus plantarum (Muller & Schultz, 1993
). AHAS has homology to all of these but is most closely related to POX, sharing 2930% amino acid identity. Indeed, E. coli AHAS II (EcilvGp) has previously been modelled on the crystal structure of LpPoxp (Ibdah et al., 1996
); both enzymes use the same cofactors, substrate and initial catalytic step and E. coli POX has been found to have a low AHAS activity (Chang & Cronan, 1988
). Alignment of the amino acid sequences of AHAS from S. cerevisiae and E. coli (AHAS II) with that of L. plantarum POX (LpPoxp) revealed that the aspartate residue that is altered by the thi1 mutation is conserved between these three sequences; D176 of yeast AHAS aligns with D84 of AHAS II of E. coli and with D96 of L. plantarum POX (Fig. 5
). Moreover, in a trans-kingdom comparison of AHAS sequences from eubacteria, archaea and eukaryotes (Chipman et al., 1998
) this aspartate was conserved in eight out of the eleven polypeptides, with an identical substitution by threonine in the other three.
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To try to gain more information about the structural effects of the thi1 mutation we used the RasMol program (Sayle & Milner-White, 1995 ), taking the LpPoxp structure as a template, to model the predicted three-dimensional structures of the Scilv2p in the wild-type (D176) and mutant (D176E) forms (Fig. 6
). A loop of the TPP-binding domain, containing residues T518 and S519, was found to lie at the interface with the core domain, between the D176 residue and the two ligands TPP and FAD. This serine residue, S519 of Scilv2p, is conserved in all 11 AHAS sequences of the trans-kingdom analysis (Chipman et al., 1998
) and in LpPoxp (Fig. 5
), and lies close to the conserved methionine residue (M525 in Scilv2p) that has been implicated in determining the conformation of the bound TPP (Duggleby & Pang, 2000
); residues of Scilv2p that interact with the phosphate of TPP via a co-ordinated Mg2+ anion are D550 and N577 (Duggleby & Pang, 2000
). In the wild-type configuration residue D176 can form a hydrogen bond across the domain interface with the residue S519. However, the mutant residue E176 does not form this hydrogen bond but instead forms a salt-bridge with R151, also of the core domain. In wild-type AHAS, R151 forms a hydrogen bond with the backbone oxygen of T518 but this interaction probably does not occur in the mutant because of the interference by E176. Thus the thi1 mutation, which substitutes a glutamate for an aspartate residue at position 176, is likely to break the hydrogen-bonding network across this domain interface of the monomer, ultimately reducing the binding affinity of AHAS enzyme for the TPP ligand.
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ACKNOWLEDGEMENTS |
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Received 30 April 2001;
accepted 29 May 2001.
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