Plasticity in the purine-thiamine metabolic network of Salmonella

In Salmonella enterica, 5-aminoimidazole ribonucleotide (AIR) is the precursor of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) pyrophosphate moiety of thiamine and the last intermediate in the common HMP/purine biosynthetic pathway. AIR is synthesized de novo via five reactions catalyzed by the purF, -D, -T, -G, and -I gene products. In vivo genetic analysis demonstrated that in the absence of these gene products AIR can be generated if (i) methionine and lysine are in the growth medium, (ii) PurC is functional, and (iii) 5-amino-4-imidazolecarboxamide ribotide (AICAR) has accumulated. This study provides evidence that the five steps of the common HMP/purine biosynthetic pathway can be bypassed in the synthesis of AIR and thus demonstrates that thiamine synthesis can be uncoupled from the early purine biosynthetic pathway in bacteria.     

Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium.

The synthesis of the pyrimidine moiety of thiamine (vitamin B1) shares five reactions with the de novo purine biosynthetic pathway. Aminoimidazole ribotide (AIR) is the last common intermediate before the two pathways diverge. Evidence for the existence of a new pathway to the pyrimidine which bypasses the de novo purine biosynthetic pathway is reported here. This pathway is only expressed under anaerobic growth conditions and is denoted alternative pyrimidine biosynthesis or APB. Labeling studies are consistent with pantothenate being a precursor to the pyrimidine moiety of thiamine that is synthesized by the APB pathway. The APB pathway is independent of the alternative purF function which was proposed previously (D. M. Downs and J. R. Roth, J. Bacteriol. 173:6597-6604, 1991). The alternative purF function is shown here to be affected by temperature and exogenous pantothenate. Although the evidence suggests that the APB pathway is separate from the alternative purF function, the relationship between this function and the APB pathway is not yet clear.     

Genetic Analysis of Metabolic Crosstalk and Its Impact on Thiamine Synthesis in Salmonella Typhimurium

The first five steps in de novo purine biosynthesis are involved in the formation of the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine. We show here that the first enzyme in de novo purine biosynthesis, PurF, is required for thiamine synthesis during aerobic growth on some but not other carbon sources. We show that PurF-independent thiamine synthesis depends on the recently described alternative pyrimidine biosynthetic (APB) pathway. Null mutations in zwf (encoding glucose-6-P dehydogenase), gnd (encoding gluconate-6-P dehydrogenase), purE (encoding aminoimidazole ribo-nucleotide carboxylase), and purR (encoding a regulator of gene expression) were found to affect the function of the APB pathway. A model is presented to account for the involvement of these gene products in thiamine biosynthesis via the APB pathway. Results presented herein demonstrate that function of the APB pathway can be prevented either by blocking intermediate formation or by diverting intermediate(s) from the pathway. Strong genetic evidence supports the conclusion that aminoimidazole ribotide (AIR) is an intermediate in the APB pathway.     

The apbE Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in Salmonella typhimurium

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S. typhimurium chromosome. Two significant phenotypes associated with lesions in this locus (apbE) were identified. First, apbE purF double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with apbE mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.     

Synthesis of thiamine in Salmonella typhimurium independent of the purF function.

In Salmonella typhimurium, the first five steps in purine biosynthesis also serve as the first steps in the biosynthesis of the pyrimidine moiety of thiamine (vitamin B1). Strains with null mutations of the first gene of purine-thiamine synthesis (purF) can, under some circumstances, grow without thiamine. This suggests the existence of an alternative pathway to thiamine that can function without the purF protein. To demonstrate the nature and map position of the purF mutations corrected, a fine-structure genetic map of the purF gene was made. The map allows identification of deletion mutations that remove virtually all of the purF gene, as defined by mutations. We describe conditions and mutations (panR) which allow B1 synthesis appears to require enzymes which act mutants lacking purF function. The alternative route of B1 synthesis appears to require enzymes which act subsequent to the purF enzyme in the purine pathway.     

1-methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism

In Salmonella enterica serovar Typhimurium a mutation in the purF gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a purF mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from S. enterica specific for leucine, proline, and arginine contain 1-methylguanosine (m(1)G37) adjacent to and 3' of the anticodon (position 37). The formation of m(1)G37 is catalyzed by the enzyme tRNA(m(1)G37)methyltransferase, which is encoded by the trmD gene. Mutations in this gene, which result in an m(1)G37 deficiency in the tRNA, in a purF mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m(1)G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m(1)G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.     

Biosynthesis of the pyrimidine moiety of thiamine. A new route of pyrimidine biosynthesis involving purine intermediates

1. The pattern of distribution on the purine pathway of mutants of Salmonella typhimurium LT2 that had the double growth requirement for a purine plus the pyrimidine moiety of thiamine (ath mutants) indicated that purines and the pyrimidine moiety of thiamine share the early part of their biosynthetic pathways, and that 4-aminoimidazole ribonucleotide (AIR) is the last common intermediate. Two mutants that at first appeared anomalous were further investigated and found not to affect this deduction. 2. The ribonucleoside form of AIR (AIR(s)) satisfied the requirements both for a purine and for the pyrimidine moiety of thiamine of an ath mutant. 3. Methionine was required for the conversion of AIR into the pyrimidine moiety. 4. Radioactive AIR(s) was converted into radioactive pyrimidine moiety by an ath mutant without significant dilution of specific radioactivity. 5. Possible mechanisms for pyrimidine-moiety biosynthesis from AIR are discussed.     

Anthranilate synthase can generate sufficient phosphoribosyl amine for thiamine synthesis in Salmonella enterica

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.     

apbA, a new genetic locus involved in thiamine biosynthesis in Salmonella typhimurium.

In Salmonella typhimurium, the synthesis of the pyrimidine moiety of thiamine can occur by utilization of the first five steps in de novo purine biosynthesis or independently of the pur genes through the alternative pyrimidine biosynthetic, or APB, pathway (D. M. Downs, J. Bacteriol. 174:1515-1521, 1992). We have isolated the first mutations defective in the APB pathway. These mutations define the apbA locus and map at 10.5 min on the S. typhimurium chromosome. We have cloned and sequenced the apbA gene and found it to encode a 32-kDa polypeptide whose sequence predicts an NAD/flavin adenine dinucleotide-binding pocket in the protein. The phenotypes of apbA mutants suggest that, under some conditions, the APB pathway is the sole source of the pyrimidine moiety of thiamine in wild-type S. typhimurium, and furthermore, the pur genetic background of the strain influences whether this pathway can function under aerobic and/or anaerobic growth conditions.     

Lesions in gshA (Encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar typhimurium LT2

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.     

Altered pathway routing in a class of Salmonella enterica serovar Typhimurium mutants defective in aminoimidazole ribonucleotide synthetase

In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K(m) of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V(max) was decreased 2.5-fold. The K(m) for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.     

Complex Metabolic Phenotypes Caused by a Mutation in yjgF,Encoding a Member of the Highly Conserved YER057c/YjgF Family of Proteins

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.The increasing number of completed genome sequences has resulted in the identification of new families of hypothetical proteins whose function has yet to be established. The lack of existing mutants defective in these conserved proteins suggests novel, complex, or subtle phenotypes. Through our work on thiamine synthesis in Salmonella typhimurium, we have isolated mutants defective in the recently identified YER057c/YjgF protein family. Our data suggest that defects in this protein result in complex phenotypes involving thiamine and isoleucine biosynthesis.Thiamine pyrophosphate (TPP) serves as an essential cofactor for a number of metabolic reactions involving the removal or transfer of C₂ units. Despite the important role of TPP in cellular metabolism, its synthesis and regulation are not well understood in any organism. TPP is formed from two precursors, 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (THZ-P) and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP). These compounds are joined and subsequently phosphorylated as shown in Fig. ​Fig.1A.1A. Although many of the enzymatic steps in both the THZ-P and HMP-PP pathways have not been clearly defined, the major precursor molecules for both of these compounds have been determined by labeling studies (17, 20, 28, 29). In particular, the purine pathway intermediate, aminoimidazole ribotide (AIR), has been shown to provide all of the atoms in HMP (28, 50, 51).Open in a separate windowFIG. 1Pathway schematics. (A) Biosynthetic pathway for TPP. The involvement of the purine pathway in HMP-PP synthesis is shown with structural intermediates prior to the AIR branch point. Arrows denoted with dotted lines represent proposed steps. Reactions involved in the conversion of AIR to HMP-PP and in the synthesis of THZ-P have not been clearly defined. Genes whose products are required for selected reactions are indicated next to the relevant arrows. Abbreviations: R-P, ribose-5-phosphate, PRPP, phosphoribosylpyrophosphate. (B) Biosynthetic pathways for the branched-chain amino acids isoleucine and valine. Enzymes that catalyze specific steps are as follows: 1, aspartate transaminase; 2, 3, and 4, aspartate kinases I, II, and III, respectively; 5, aspartate semialdehyde dehydrogenase; 6 and 7, homoserine dehydrogenases I and II, respectively; 8, homoserine kinase; 9, threonine synthase; 10, threonine deaminase; 11 and 12, acetohydroxy acid synthases I and II, respectively; 13, acetohydroxy acid isomeroreductase; 14, dihydroxy acid dehydratase; 15, transaminase B; 16, transaminase C. OAA, oxaloacetic acid.Although the involvement of the purine pathway in the synthesis of HMP is clear, there is substantial genetic and biochemical evidence indicating that the first enzyme of the purine pathway, phosphoribosylpyrophosphate amidotransferase (PurF) (EC 2.4.2.14), is not required for HMP synthesis in S. typhimurium under all conditions. Mutants defective in purF are able to grow in the absence of thiamine when glucose is used as a carbon source if pantothenate is also supplied in the medium (23). Similarly, purF mutants do not require thiamine when grown on a number of nonglucose carbon sources, such as gluconate or ribose (54). The pathway responsible for synthesis of HMP independent of the PurF enzyme has been defined as the alternative pyrimidine biosynthetic (APB) pathway (21, 54); recent biochemical data suggest that phosphoribosylamine (PRA), or a derivative, is an intermediate in this pathway (24).Significant progress in our understanding of the APB pathway has been made by the isolation and characterization of mutants unable to synthesize thiamine in a purF background. One class of mutants, designated apbA, was defective in a pantothenate biosynthetic enzyme (ketopantoate reductase [PanE]) (32, 33), consistent with previous results implicating a role for pantothenate in PurF-independent thiamine synthesis (23). A second class of these mutants was defective in the oxidative pentose phosphate pathway, affecting either glucose-6-phosphate dehydrogenase (Zwf) or gluconate-6-phosphate dehydrogenase (Gnd) (25, 54). Addition of ribose-5-phosphate (ribose-5-P) restored function of the APB pathway in these mutants, suggesting that the role of these enzymes in HMP synthesis was to supply ribose-5-P. These results led to the model shown in Fig. ​Fig.1A1A which implicates ribose-5-P and an amine donor as precursors to PRA. Repeated attempts have failed to identify either the predicted PRA-forming activity or mutants defective in this step (27). There are several possible explanations for this. It is possible that the correct substrates have not been identified and/or that the PRA-forming activity is required for another cellular function.In this report, we describe the isolation and characterization of mutations that allow function of the APB pathway in the absence of the pentose phosphate pathway. These mutations were found to disrupt a previously uncharacterized open reading frame (ORF) encoding a hypothetical 13.5-kDa protein. We have designated this gene yjgF based on homology to the respective ORF in Escherichia coli. The YjgF protein belongs to the YER057c/YjgF protein family, a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms. Characterization of these mutants revealed that they also were sensitive to the presence of serine in the medium, exhibiting a requirement for isoleucine under this condition. The phenotypes caused by yjgF mutations suggest that the YjgF protein may be involved in regulation or function of the isoleucine biosynthetic pathway. Further, results suggest a connection between isoleucine biosynthesis and function of the APB pathway in thiamine synthesis.     

Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis.

Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways. In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis [Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We have cloned a 3.1-kb genomic S. pombe fragment which can functionally complement a thi2 mutant. The fragment maps genetically at the thi2 site, indicating that it carries thi2. As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole. The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine [Maundrell, J. Biol. Chem. 265 (1990) 10857-10864; Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 [Schweingruber et al., Genetics (1992) in press]. Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3. This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S. pombe.     

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.     

Cloning and characterization of the rpoE gene encoding an RNA polymerase sigmaE factor from the deep-sea piezophilic Shewanella violacea strain DSS12.

The rpoE gene encoding an RNA polymerase sigmaE subunit was isolated from a gamma-phage library of the deep-sea piezophilic and psychrophilic bacterium Shewanella violacea strain DSS12. Structual analysis showed that the gene organization of the fragment containing S. violacearpoE was the l-aspartate oxidase-coding gene, rpoE, rseA, rseB and rseC in that order, the same as in the case of Photobacterium profundum SS9 and Escherichia coli K-12. The cloned gene, 576 bp in length, was found to encode a protein consisting of 192 amino acid residues with a molecular mass of 21,806 Da. Amino acid alignment of the RpoE protein showed that the functional domains responsible for DNA recognition, DNA melting, core binding, and RseA interaction were highly conserved. We purified hexahistidine-fused RpoE protein by constructing an overexpression plasmid. Core-binding analysis revealed that the cloned RpoE protein has the ability to bind with core RNA polymerase as a sigma factor.     

The panE Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to apbA in Salmonella typhimurium

In Salmonella typhimurium, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. The apbA gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. Petersen, J. Bacteriol. 176:4858–4864, 1994). Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid. This activity had previously been associated with the pantothenate biosynthetic gene panE. Although previous reports placed panE at 87 min on the Escherichia coli chromosome, we show herein that apbA and panE are allelic and map to 10 min on both the S. typhimurium and E. coli chromosomes. Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway.     

Vitamin B1 de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine

Vitamin B1 (thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of thiamine precursors.