Thi20, a remarkable enzyme from Saccharomyces cerevisiae with dual thiamin biosynthetic and degradation activities

Saccharomyces cerevisiae Thi20 is a fusion protein with homology to Bacillus subtilis ThiD and TenA. The N-terminus of Thi20 has significant sequence homology to B. subtilis ThiD, while the C-terminus has homology to B. subtilis TenA. Incubation of Thi20 with thiamin reveals that it has thiaminase II activity, in addition, incubation of Thi20 with HMP (4-amino-2-methyl-5-hydroxymethylpyrimidine) and ATP reveals that it has HMP kinase and HMP-P (4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate) kinase activity. This demonstrates that Thi20 is a trifunctional protein with thiamin biosynthetic and degradative activity.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution.

The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme.     

Involvement of thiaminase II encoded by the THI20 gene in thiamin salvage of Saccharomyces cerevisiae

The physiological significance of thiaminase II, which catalyzes the hydrolysis of thiamin, has remained elusive for several decades. The C-terminal domains of THI20 family proteins (THI20/21/22) and the whole region of PET18 gene product of Saccharomyces cerevisiae are homologous to bacterial thiaminase II. On the other hand, the N-terminal domains of THI20 and THI21 encode 2-methyl-4-amino-5-hydroxymethylpyrimidine kinase and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase involved in the thiamin synthetic pathway. In this study, it was first indicated that the C-terminal domains of the THI20 family and PET18 are not required for de novo thiamin synthesis in S. cerevisiae, using a quadruple deletion strain expressing the N-terminal domain of THI20. Biochemical analysis using cell-free extracts and recombinant proteins demonstrated that yeast thiaminase II activity is exclusively encoded by THI20. It appeared that Thi20p has an affinity for the pyrimidine moiety of thiamin, and HMP produced by the thiaminase II activity is immediately phosphorylated. Thi20p was found to participate in the formation of thiamin from two synthetic antagonists, pyrithiamin and oxythiamin, by hydrolyzing both antagonists and phosphorylating HMP to give HMP pyrophosphate. Furthermore, 2-methyl-4-amino-5-aminomethylpyrimidine, a presumed naturally occurring thiamin precursor, was effectively converted to HMP by incubation with Thi20p. It is proposed that the thiaminase II activity of Thi20p is involved in the thiamin salvage pathway by catalyzing the hydrolysis of HMP precursors in S. cerevisiae.     

Enzymatic and structural characterization of an archaeal thiamin phosphate synthase

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme–product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.     

Structural characterization of the regulatory proteins TenA and TenI from Bacillus subtilis and identification of TenA as a thiaminase II

Bacillus subtilis gene products TenA and TenI have been implicated in regulating the production of extracellular proteases, but their role in the regulation process remains unclear. The structural characterization of these proteins was undertaken to help provide insight into their function. We have determined the structure of TenA alone and in complex with 4-amino-2-methyl-5-hydroxymethylpyrimidine, and we demonstrate that TenA is a thiaminase II. The TenA structure suggests that the degradation of thiamin by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I. Three active-site residues, Asp44, Cys135, and Glu205, are likely involved in substrate binding and catalysis based on the enzyme/product complex structure and the conservation of these residues within TenA sequences. We have also determined the structure of TenI. Although TenI shows significant structural homology to thiamin phosphate synthase, it has no known enzymatic function. The structure suggests that TenI is unable to bind thiamin phosphate, largely resulting from the presence of leucine at position 119, while the corresponding residue in thiamin phosphate synthase is glycine.     

Determination of the genetic, molecular, and biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1

2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase/thiamin monophosphate pyrophosphorylase (HMPPK/TMPPase) is a key enzyme involved in thiamin biosynthesis. A candidate HMPPK/TMPPase gene identified in the Arabidopsis genome complemented the thiamin auxotrophy of the th1 mutant, thus proving that the th1 locus corresponds to the structural gene for the HMPPK/TMPPase. Sequence comparisons between the wild-type HMPPK/TMPPase gene and the th1-201 mutant allele identified a single point mutation that caused the substitution of a phenylalanine for a conserved serine residue in the HMPPK domain. Functional analyses of the mutant HMPPK/TMPPase in Escherichia coli revealed that the amino acid substitution in the HMPPK domain of mutant enzyme resulted in a conformational change that severely compromised both activities of the bifunctional enzyme. Studies were also performed to identify the chloroplast as the specific subcellular locale of the Arabidopsis HMPPK/TMPPase.     

Recent progress in understanding thiamin biosynthesis and its genetic regulation in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to synthesize thiamin pyrophosphate (TPP) de novo, which involves the independent formation of two ring structures, 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-β-hydroxyethylthiazole, in the early steps. In addition, this organism can efficiently utilize thiamin from the extracellular environment to produce TPP. Nineteen genes involved in the synthesis of TPP and the utilization of thiamin (THI genes) have been identified, and the function of several THI genes has been elucidated. All THI genes participating in the synthesis of the pyrimidine unit belong to multigene families. It is also intriguing that some thiamin biosynthetic proteins are composed of two distinct domains or form an enzyme complex. The expression of THI genes is coordinately induced in response to thiamin starvation. It is likely that the induction of THI genes is activated by a positive regulatory factor complex and that the protein–protein interaction among the factors is disturbed by TPP. Thiamin-hyperproducing yeast and fermented food containing a high content of thiamin are expected to be available in the future based on the progress in understanding thiamin biosynthesis and its genetic regulation in S. cerevisiae.     

Transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine in Saccharomyces cerevisiae

The transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) was studied in resting cells of Saccharomyces cerevisiae. Hydroxymethylpyrimidine uptake was an energy- and temperature-dependent process which has an optimal pH at 4.5. The apparent Km for hydroxymethylpyrimidine uptake was 0.37 microM, and the uptake was inhibited by 2-methyl-4-amino-5-aminomethylpyrimidine, thiamin and pyrithiamin. Furthermore, hydroxymethylpyrimidine uptake was inhibited by 4-azido-2-nitrobenzoylthiamin, a specific and irreversible inhibitor of the yeast thiamin transport system and it was greatly impaired in the thiamin transport mutant of S. cerevisiae. Thus, hydroxymethylpyrimidine is taken up by a common transport system with thiamin in S. cerevisiae, but in contrast to thiamin transport, accumulated hydroxymethylpyrimidine is released from yeast cells showing an overshoot phenomenon.     

Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism

Thiamin phosphate synthase catalyzes the coupling of 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate (Thz-P) and 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) to give thiamin phosphate. In this paper, we demonstrate that 4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine pyrophosphate (CF(3)-HMP-PP) is a very poor substrate [k(cat)(CH(3)) > 7800k(cat)(CF(3))] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH(3)O-HMP-PP) is a good substrate [k(cat)(OCH(3)) > 2.8k(cat)(CH(3))] for the enzyme. We also demonstrate that the enzyme catalyzes positional isotope exchange. These observations are consistent with a dissociative mechanism (S(N)1 like) for thiamin phosphate synthase in which the pyrimidine pyrophosphate dissociates to give a reactive pyrimidine intermediate which is then trapped by the thiazole moiety.     

Structure of the thiazole biosynthetic enzyme THI1 from Arabidopsis thaliana

Thiamin pyrophosphate is an essential coenzyme in all organisms that depend on fermentation, respiration or photosynthesis to produce ATP. It is synthesized through two independent biosynthetic routes: one for the synthesis of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (pyrimidine moiety) and another for the synthesis of 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate (thiazole moiety). Herein, we will describe the three-dimensional structure of THI1 protein from Arabidopsis thaliana determined by single wavelength anomalous diffraction to 1.6A resolution. The protein was produced using heterologous expression in bacteria, unexpectedly bound to 2-carboxylate-4-methyl-5-beta-(ethyl adenosine 5-diphosphate) thiazole, a potential intermediate of the thiazole biosynthesis in Eukaryotes. THI1 has a topology similar to dinucleotide binding domains and although details concerning its function are unknown, this work provides new clues about the thiazole biosynthesis in Eukaryotes.     

Some properties of a Saccharomyces cerevisiae mutant resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole

A mutant of Saccharomyces cerevisiae highly resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole (2-aminohydroxyethylthiazole), an antimetabolite of 4-methyl-5-beta-hydroxyethylthiazole (hydroxyethylthiazole), has been isolated. Its resistance to 2-aminohydroxyethylthiazole was about 10(4) times that of the sensitive parent strain. The amount of thiamin synthesized in the cells of the resistant strain grown in minimal medium was less than half of that of the sensitive strain. The ability to synthesize thiamin from 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and hydroxyethylthiazole in the resistant strain was low compared with that of the sensitive strain. These results were found to be due to a deficiency of hydroxyethylthiazole kinase in the resistant strain: in sonic extracts of cells the enzyme activity was only 0.67% of that of the sensitive strain. Although the cells of the sensitive strain could accumulate exogenous hydroxyethylthiazole in the form of hydroxyethylthiazole monophosphate, no significant uptake of hydroxyethylthiazole by the cells of the resistant strain was observed. The possibilities that 2-aminohydroxyethylthiazole monophosphate may be the actual inhibitor of the growth of Saccharomyces cerevisiae, and that hydroxyethylthiazole may not be involved in the pathway of de novo synthesis of thiamin via hydroxyethylthiazole monophosphate, are discussed.     

Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase

Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme-substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures.     

Crystal structure of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 A resolution

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole (Thz). This enzyme is a salvage enzyme in the thiamin biosynthetic pathway and enables the cell to use recycled Thz as an alternative to its synthesis from 1-deoxy-D-xylulose-5-phosphate, cysteine, and tyrosine. The structure of ThiK in the rhombohedral crystal form has been determined to 1.5 A resolution and refined to a final R-factor of 21. 6% (R-free 25.1%). The structures of the enzyme/Thz complex and the enzyme/Thz-phosphate/ATP complex have also been determined. ThiK is a trimer of identical subunits. Each subunit contains a large nine-stranded central beta-sheet flanked by helices. The overall fold is similar to that of ribokinase and adenosine kinase, although sequence similarity is not immediately apparent. The area of greatest similarity occurs in the ATP-binding site where several key residues are highly conserved. Unlike adenosine kinase and ribokinase, in which the active site is located between two domains within a single subunit, the ThiK active site it formed at the interface between two subunits within the trimer. The structure of the enzyme/ATP/Thz-phosphate complex suggests that phosphate transfer occurs by an inline mechanism. Although this mechanism is similar to that proposed for both ribokinase and adenosine kinase, ThiK lacks an absolutely conserved Asp thought to be important for catalysis in the other two enzymes. Instead, ThiK has a conserved cysteine (Cys198) in this position. When this Cys is mutated to Asp, the enzymatic activity increases 10-fold. Further sequence analysis suggests that another thiamin biosynthetic enzyme (ThiD), which catalyzes the formation of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by two sequential phosphorylation reactions, belongs to the same family of small molecule kinases.     

A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae.

We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.     

The mechanism of action of bacimethrin, a naturally occurring thiamin antimetabolite

The mechanism of bacimethrin (2) toxicity has been determined. This compound is converted to 2'-methoxy-thiamin pyrophosphate (10) by the thiamin biosynthetic enzymes. Of the seven thiamin pyrophosphate utilizing enzymes in Escherichia coli, 2'-methoxy-thiamin pyrophosphate inhibits alpha-ketoglutarate dehydrogenase, transketolase, and deoxy-D-xylulose-5-phosphate synthase. Bacimethrin does not cause repression of the genes coding for the thiamin biosynthetic enzymes.     

Characterization of two kinases involved in thiamine pyrophosphate and pyridoxal phosphate biosynthesis in Bacillus subtilis: 4-amino-5-hydroxymethyl-2methylpyrimidine kinase and pyridoxal kinase

Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized. YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B. subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.     

Transport overshoot during 2-methyl-4-amino-5-hydroxymethylpyrimidine uptake by Saccharomyces cerevisiae

The transport overshoot during 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) uptake by the thiamin transport system in Saccharomyces cerevisiae was investigated. The overshoot was found to be temperature- and energy-dependent and affected by the growth phase of the yeast. The efflux system for hydroxymethylpyrimidine appeared to be more sensitive to 2,4-dinitrophenol than the influx system, resulting in the loss of the overshoot of the pyrimidine in the presence of the uncoupler. Furthermore, the overshoot did not occur after the preincubation of yeast cells with inhibitors of protein synthesis such as cycloheximide and anisomycin. These results suggest that an active efflux system for hydroxymethylpyrimidine, which is rapidly synthesized, is involved in the overshoot of this pyrimidine during its transport in S. cerevisiae.     

Hydroxyethylthiazole Uptake in Escherichia coli: General Properties and Relationship Between Uptake and Thiamine Biosynthesis

Uptake of (35)S-hydroxyethylthiazole (4-methyl-5-hydroxyethylthiazole) by Escherichia coli intact cells was studied. Hydroxyethylthiazole was taken up in the presence and absence of glucose at the same rate. The uptake was almost proportional to a hydroxyethylthiazole concentration gradient up to 0.1 mM with no tendency of saturation, and reached a steady state within 2 min. When the cells were treated with 1 mM N-ethylmaleimide, about 50% inhibition of hydroxyethylthiazole uptake was observed. Hydroxyethylthiazole uptake was stimulated by the addition of hydroxymethylpyrimidine (2-methyl-4-amino-5-hydroxymethylpyrimidine), and this effect was further enhanced in the presence of glucose. For full activation of hydroxyethylthiazole uptake, 1 muM hydroxymethylpyrimidine was necessary in the presence of glucose. The rate of hydroxyethylthiazole uptake was almost linear up to 60 min in the presence of hydroxymethylpyrimidine and glucose. Hydroxymethylpyrimidine monophosphate and its pyrophosphate could not stimulate the uptake. Thiamine and 2-amino-hydroxyethylthiazole were inhibitory on hydroxyethylthiazole uptake in the presence of hydroxymethylpyrimidine and glucose. N-ethylmaleimide and 2, 4-dinitrophenol were also inhibitory. No stimulatory effect of hydroxymethylpyrimidine on hydroxyethylthiazole uptake was observed in mutant cells lacking either thiaminephosphate pyrophosphorylase or hydroxymethylpyrimidine monophosphate kinase. The possibility of direct participation of thiamine-synthesizing enzymes in hydroxyethylthiazole uptake was discussed.     

A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis

We report the characterization of a Brassica napus cDNA clone (pBTH1) encoding a protein (BTH1) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.