Biochemical evidence for the existence of thymidylate synthase in the obligate intracellular parasite Chlamydia trachomatis.

Since eucaryotic cell-derived thymidine or thymidine nucleotides are not incorporated into Chlamydia trachomatis DNA, we hypothesized that C. trachomatis must obtain dTTP for DNA synthesis by converting dUMP to dTMP. In most cells, this reaction is catalyzed by thymidylate synthase (TS) and requires 5,10-methylenetetrahydrofolate as a cofactor. We used C. trachomatis serovar L2 and a mutant CHO K1 cell line with a genetic deficiency in folate metabolism as a host for chlamydial growth. This cell line lacks a functional dihydrofolate reductase (DHFR) gene and, as a result, is unable to carry out de novo synthesis of dTTP. C. trachomatis inclusions form normally when DHFR- cells are starved for thymidine 24 h prior to and during the course of infection. When [6-3H]uridine is used as a precursor to label C. trachomatis-infected CHO DHFR- cells, radiolabel is readily incorporated into chlamydia-specific DNA. When DNA from [6-3H]uridine-labelled infected cultures is acid hydrolyzed and subjected to high-performance liquid chromatography analysis, radiolabel is detected in thymine and cytosine nucleobases. By using the DHFR- cell line as a host and [5-3H]uridine as a precursor, we could monitor intracellular C. trachomatis TS activity simply by following the formation of tritiated water. There is a good correlation between in situ TS activity and DNA synthesis activity during the chlamydial growth cycle. In addition, both C. trachomatis-specific DNA synthesis and 3H2O release are inhibited by exogenously added 5-fluorouridine but not by 5-fluorodeoxyuridine. Finally, we demonstrated in vitro TS activity in crude extracts prepared from highly purified C. trachomatis reticulate bodies. The activity is dependent on the presence of methylenetetrahydrofolic acid and can be inhibited with 5-fluoro-dUMP. Taken together, these results indicate that C. trachomatis contains a TS for the synthesis of dTMP.     

Biosynthesis of methanopterin

The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. [methylene-2H]-6-(Hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from [methyl-2H3]methionine, as confirmed by the incorporation of two C2H3 groups into 6-ethyl-7-methylpterin, a pterin-containing fragment derived from methanopterin. Cells grown in the presence of [methylene-2H]-6-(hydroxymethyl)pterin, [ethyl-2H4]-6-[1 (RS)-hydroxyethyl]pterin, [methyl-2H3]-6- (hydroxymethyl)-7-methylpterin, [ethyl-2H4, methyl-2H3]-6-[1 (RS)-hydroxyethyl]-7-methylpterin, and [1-ethyl-3H]-6-[1 (RS)-hydroxyethyl]-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic [methylene-3H]-7,8-H2-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H2-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.     

Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

Calditol tetraether lipids of the archaebacterium Sulfolobus solfataricus. Biosynthetic studies.

Lipids from the archaebacterium Sulfolobus solfataricus are based on 72-membered macrocyclic tetraethers made up from two C40 diol units differently cyclized and either two glycerol moieties or one glycerol moiety and a unique branched-chain nonitol named calditol (glycerodialkylnonitol tetraethers, GDNTs). To elucidate the biosynthesis of calditol and related tetraethers, labelled precursors, [U-14C,1(3)-3H]glycerol, [U-14C,2-3H]glycerol, D-[1-14C,6-3H]glucose, D-[6-14C,1-3H]glucose, D-[1-14C,2-3H]glucose, D-[1-14C,6-3H]fructose and D-[1-14C]galactose, were fed to S. solfataricus. Without regard to stereochemistry or phosphorylation, incorporation experiments provided evidence that the biosynthesis of calditol occurs via an aldolic condensation between dihydroxyacetone and fructose, through a 2-oxo derivative of calditol as an intermediate. The latter is in turn reduced and then alkylated to yield the GDNTs. The biogenetic origins of both glycerol and C40 isoprenoid moieties of GDNTs are also discussed.     

A method for the determination of total,free, and 5-fluorodeoxyuridylate-bound thymidylate synthase in cell extracts

A radiochemical assay for thymidylate synthase (EC 2.1.1.45, dTMP synthase), which permits the accurate determination of total, free, and 5-fluoro-2′-deoxyuridylate (FdUMP)-bound enzyme in cells exposed to the 5-fluoropyrimidine anticancer agents, is described. The total intracellular concentrations of dTMP synthase (free plus FdUMP-bound enzyme) in extracts from CCRF-CEM leukemic cells incubated with 5-fluoro-2′-deoxyuridine were determined following dissociation of the covalent dTMP synthase-5,10-methylenetetrahydrofolate-FdUMP ternary complex in the presence of the substrate, 2′-deoxyuridine-5′-monophosphate. The addition of substrate prevented reformation of the ternary complex during the dissociation procedure, and allowed complete recovery of FdUMP binding sites in cells exposed to a high concentration of 5-fluoro-2′-deoxyuridine. After removal of the substrate by charcoal adsorption, the concentration of total FdUMP binding sites was determined by titration of the enzyme with a saturating concentration of [6-³H]FdUMP and 5,10-methylenetetrahydrofolate. The concentration of FdUMP-bound dTMP synthase was then calculated as the difference between the total and free (without prior ternary complex disruption) enzyme values. The high sensitivity of this assay coupled with its ability to accurately quantitate both free and FdUMP-bound dTMP synthase in cells exposed to a wide range of fluoropyrimidine concentrations should make it useful for a variety of experimental and clinical studies.     

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.     

D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH.

The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea. In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion. In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes. A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate. Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M. thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.     

Naphthalene-fused (α-alkoxycarbonyl)methylene-γ-butyrolactones: antiproliferative activity and binding to bovine serum albumin and DNA

A naphthalene-fused (α-alkoxycarbonyl)methylene-γ-butyrolactone (methyl 2-[7-hydroxy-2-oxonaphtho[1,2-b]furan-3(2H)-yliden]acetate) has been prepared as a representative compound of a potential class of cytotoxic agents. In vitro cytotoxicity has been evaluated against HCT-15 colon and MCF-7 breast cancer cells and IC(50) was 64-66 μM, causing morphological changes in cells, such as loss of adhesion, rounding, cell shrinkage, and detachment from the substratum. The binding constant K of the complex between the naphthyl lactone with bovine serum albumin (8 × 10(3) M(-1)) suggests a minor change in protein folding. The K of the binding with DNA (1.06 × 10(4) M(-1)) suggests nonspecific electrostatic interactions with DNA and this was confirmed by melting point data (Tm<0.6 °C). Therefore, naphthalene-fused (α-alkoxycarbonyl)methylene-γ-butyrolactone should not be able to intercalate with DNA but its interaction should occur at the level of DNA surface.     

Suppressor of deoxythmidine monophosphate uptake in Saccharomyces cerevisiae.

Although yeast cannot normally incorporate exogenous deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid, mutants able to do so have been isolated. We have characterized a recessive suppressor of dTMP uptake (sot1) that prevents strains carrying either tup1, tup2, or tup4 from growing on selective medium. The sot1 mutation maps between rad1 and the centromere of chromosome XVI, and is unlinked to any of the tup mutations. The sot1 mutation does not suppress the other pleiotropic effects of the tup1 mutant, notably the lack of mating of tup1 MATalpha strains. The sot1 mutation specifically blocks the uptake of dTMP into tup strains. After growing a sot1 strain in medium containing [3H]dTMP, we showed that the medium still contained more than 90% of the original [3H]dTMP and that this medium could support the incorporation of [3H]dTMP by a tup2 strain. Therefore, sot1 strains do not degrade dTMP in the medium. The sot1 mutation had no effect on the uptake of other nutrients essential for growth, including several amino acids, adenine, and uracil.     

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA synthesis in toluene-treated bacteriophage-infected minicells or Bacillus subtilis.

Bateriophage (phi29, SPP1, or SPO1)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [3H]dTTP into a trichloroacetic acid-precipitable form. The [3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2 . 10(5)). Short exposure of the DNA molecules containing the incorporated [3H]dTMP to Escherichia coli exonuclease III results in over 90% of the E13H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil.     

Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.     

2-(7,13-Dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-3H]taurine and [1-14C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of 3H and 14C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

Si-face stereospecificity at C5 of coenzyme F420 for F420H2 oxidase from methanogenic Archaea as determined by mass spectrometry

Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5 dihydro-coenzyme F420 is formed with a prochiral centre at C5. All the coenzyme F420-dependent enzymes investigated to date have been shown to be Si-face stereospecific with respect to C5 of the deazaflavin, despite most F420-dependent enzymes being unrelated phylogenetically. In this study, we report that the recently discovered F420H2 oxidase from methanogenic Archaea is also Si-face stereospecific. The enzyme was found to catalyse the oxidation of (5S)-[5-2H1]F420H2 with O2 to [5-1H]F420 rather than to [5-2H]F420 as determined by MALDI-TOF MS. (5S)-[5-2H1]F420H2 was generated by stereospecific enzymatic reduction of F420 with (14a-2H2)-[14a-2H2] methylenetetrahydromethanopterin.     

Immobilization antigens from Tetrahymena thermophila are glycosyl-phosphatidylinositol-linked proteins.

We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3H]ethanolamine or [3H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide electrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-linked glycosyl-phosphatidylinositol anchors.     

Simple separation of tritiated water and [3H]deoxyuridine from [5-3H]deoxyuridine 5'-monophosphate in the thymidylate synthase assay

A simple micromethod was developed for the accurate measurement of the activity of dTMP synthase in rat liver crude extracts. The reaction product of dTMP synthase activity assay, i.e., tritiated water, generated by the release of tritium from carbon-5 of [5-3H]deoxyuridine 5'-monophosphate (dUMP), was separated simply by 100% KOH absorption from [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while [3H]dUrd remained in the bottom of vessels after absorption of the substrate, [5-3H]dUMP, from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extract of rat liver, the specific activities of dTMP synthase and dUMP phosphatase were 0.092 +/- 0.002 and 0.351 +/- 0.013 nmol/h/mg protein, respectively. This method was also adapted for dTMP synthase assay in crude extracts of rat hepatoma 3924A. The major advantages of this procedure are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity in crude extracts, one-step separation of 3H2O, high sensitivity (with a limit of detection of 10 pmol of 3H2O production), high reproducibility (less than +/- 4.3%), and capability to measure activity in small amounts of sample (30-45 micrograms protein).     

Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable alpha-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for alpha-linked galactosides, which are optimally hydrolyzed at pH 5 and 90 degrees C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable alpha-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.     

S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus. Purification, molecular properties and studies on the covalently bound pyruvate.

S-Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min-1 (mg protein)-1 and the overall yield 8%. The enzyme is thermophilic with an optimum at 75 degrees C, is thermostable, and does not require divalent cations or putrescine for activity. It has a molecular mass of 32 kDa, and appears to be a monomeric protein. S-Adenosylmethionine decarboxylase from S. solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time-dependent process by NaCNBH3, in the presence of both the substrate and the product. Incubation with decarboxylated S-adenosyl[Me-3H]methionine and NaCNBH3 resulted in the labeling of the protein at the active site.