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The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2(1), with unit cell dimensions a = 102.4 A, b = 116.5 A, c = 115.8 A, and beta = 103.5. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 A3/ Da. The crystals diffract to at least 2.3 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.

The one-carbon metabolism enzymes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) can be found on a single trifunctional protein in the eukaryotes examined. The one exception is in spinach leaves where 10-formyltetrahydrofolate synthetase is monofunctional (Nour, J. M., and Rabinowitz, J. C. (1991) J. Biol. Chem. 266, 18363-18369). In the prokaryotes examined, 10-formyltetrahydrofolate synthetase is either absent or is monofunctional. A cDNA clone encoding spinach leaf 10-formyltetrahydrofolate synthetase was isolated through the use of antibodies to the purified enzyme. This clone had an open reading frame of 1914 base pairs and encoded for a protein containing 636 amino acids with a calculated M(r) of 67,727. The percentage identity between spinach 10-formyltetrahydrofolate synthetase and the synthetase domains in the four trifunctional eukaryotic enzymes and the two monofunctional prokaryotic enzymes that have been cloned and sequenced was: 64.9% human, 63.8% rat, 55.6% yeast cytoplasm, 53.8% yeast mitochondria, 47.8% Clostridium acidi-urici, and 47.9% Clostridium thermoaceticum. Clearly the spinach monofunctional protein had greatest homology with the mammalian proteins. The spinach protein is longer than the two other monofunctional prokaryotic proteins. Possible reasons for this are presented. The codon usage and the putative translation initiation sites are examined and compared with other spinach proteins.

An assay for Clostridium pasteurianum apoferredoxin messenger ribonucleic acid (mRNA) was developed, based on the synthesis of the protein in vitro. Quantitation of apoferredoxin synthesis was accomplished by trypsinization of the cell-free incubation labeled with 3H- or 14C-labeled amino acids, separation of the products by SDS-urea polyacrylamide gel electrophoresis, and excising and counting the NCS-solubilized gel band corresponding to the unique 52-amino acid tryptic peptide derived from apoferredoxin. Its synthesis was shown to be RNA dependent, and was optimized with respect to several parameters of the in vitro protein-synthesizing system. The specificity of the assay was examined with RNA from Clostridium acidi urici, a related species the ferredoxin of which does not yield the 52-amino acid tryptic peptide, and by the use of [3H]leucine, which is not present in C. pasteurianum apoferredoxin. By these methods, the overestimation of apoferredoxin synthesis due to the comigration of fragments from other in vitro products with the legitimate apoferredoxin-derived peptide could be accounted for. The apoferredoxin mRNA was partially purified by the sequential zonal sucrose gradient centrifugation of total RNA followed by Sephadex G-200 chromatography of the enriched RNA, after which a fraction was obtained in which apoferredoxin mRNA was 20-fold enriched. The enriched RNA fraction can now be used for further purification of the apoferredoxin-coding sequences by cloning procedures.

We have constructed a library of Clostridium pasteurianum DNA cloned in the plasmid pBR322. Based on the known amino acid sequence for C. pasteurianum ferredoxin, a 64-fold degenerate heptadecanucleotide pool was synthesized. This mixed probe hybridized to two clones which were shown to contain greater than 6 kilobase pairs of the same genomic DNA. Sequence analysis of a common Sau3A1 0.6-kilobase-pair fragment revealed that it contains the information for the apoferredoxin structural gene. According to the DNA sequence, the only post-translational processing of this small apoprotein is the hydrolysis of the initiator methionine. Putative transcription and translation start and stop signals are present within the sequence.

We report here the isolation of a prokaryotic RNA polymerase that shows pronounced template specificity. The enzyme from Clostridium acidi-urici is highly active on DNA templates isolated from phage that infect Gram-positive organisms and is essentially inactive at either high or low ionic strength on DNA from phage associated with Gram-negative bacteria. The enzyme is also unique among RNA polymerases isolated from wild type bacteria in being highly resistant to inhibition by rifampicin. These properties are characteristic of the enzyme present in several independently isolated strains of C. acidi-urici. However, RNA polymerase present in other clostridial species resembles the enzyme present in Bacillus subtilis in sensitivity to rifampicin and template specificity.

Tyrosyl or other aromatic residues generally occur in two conserved positions in the peptide chain of clostridial-type ferredoxins and have been implicated in the electron transfer function of these iron-sulfur proteins. We have prepared and determined some of the properties of a derivative of Clostridium acidi-urici ferredoxin, [Leu(2)]-ferredoxin, in which a leucyl residue has been substituted for the tyrosyl residue in position 2 from the amino terminus. [Leu(2)]-ferredoxin is fully active as an electron carrier in two biological assays, the phosphoroclastic enzyme system and the ferredoxin-dependent reduction of cytochrome c in the presence of ferredoxin-TPN reductase and TPNH. Quantitative electron paramagnetic resonance experiments indicate that [Leu(2)]-ferredoxin accepts nearly two electrons upon enzymatic reduction by pyruvate-ferredoxin oxidoreductase and an excess of pyruvate. If electron transfer to an iron-sulfur cluster is the rate-limiting step in the assays used, and if the rate of electron transfer through Tyr(30) is not much faster than through Tyr(2), these results indicate that the primary pathway of electron transfer in clostridial-type ferredoxins is not via Tyr or other aromatic amino-acid residues. The syntheses of other ferredoxin derivatives with amino-acid substitutions or deletions in positions 1 and 2 indicate that a large bulky residue, but not necessarily an aromatic residue, is needed in position 2 for the stability of this ferredoxin. The residue in position 2, therefore, appears to act as a hydrophobic shield for an iron-sulfur cluster.

Six strains of Clostridium acidiurici and three strains of C. cylindrosporum were isolated from soil samples by enrichment culture with uric acid as the source of carbon, nitrogen, and energy. The newly isolated strains were characterized by their spore morphology and the amounts of glycine and formate formed by the fermentation of uric acid. The strains were easily identified as belonging to one species or the other on the basis of spore morphology and formate production. The crystal properties and spectra of the native ferredoxins of all the strains isolated and the amino acid composition and partial carboxy-terminal sequence of all their apoferredoxins were determined. All the ferredoxins were tested for cross-reactivity with antiserum to C. acidiurici ferredoxin by microcomplement fixation. Five of the six C. acidiurici strains, which had ferredoxins with amino acid compositions identical to that from C. acidiurici, also showed immunological identity (immunological distance = 0.0). These results suggest sequence identity. The one strain with a different amino acid composition failed to show complete cross-reactivity. Two of the three C. cylindrosporum strains have ferredoxin amino acid compositions identical to that from C. cylindrosporum. The third strain had a minimum of five differences in sequence. All C. cylindrosporum strains had ferredoxins that differed considerably from C. acidiurici strains (minimum of eight to nine differences), and none of these ferredoxins cross-reacted with antisera to C. acidiurici ferredoxin. Antisera were prepared to formyltetrahydrofolate synthetase from C. acidiurici and C. cylindrosporum, and all possible comparisons were made by using immunodiffusion and microcomplement fixation. There is more intraspecies variation in the synthetases than in the ferredoxins; however, the results suggest considerable interspecies differences in both proteins. These results suggest a low degree of genomic relatedness between the two species, which contrasts sharply with their apparent high degree of phenotypic similarity.

The semisynthetic syntheses and some properties of derivatives of Clostridium acidi-urici ferredoxin that contain amino acid deletions or replacements in the peptide chain are described. All 16 stable derivatives prepared, with the exception of [Trp2]ferredoxin, were fully active as electron carriers in the two enzymatic assay systems tested: the phosphoroclastic system and the ferrodoxin-dependent reduction of cytochrome c. E1Trp1]Ferredoxin had 70% of the activity of native ferredoxin in both assay systems. The stability in aerobic solution of [Ala1]ferredoxin, which had had its natural alanyl NH2-terminal residue removed and then replaced chemically, is the same as that of the native ferrodoxin (half-life of approximately 54 days). The relative stabilities of derivatives with a replacement or deletion of the NH2-terminal residue are as follows: [Ala1]- greater than or equal to [Phe1]-, [Lys1]-, [ Pro1]-, [Leu1]- greater than [Met1]- greater than [Gly1]- greater than [Glu1]- greater than des-Ala1-ferrodoxin. The data indicate that a large bulky residue, but not a negatively charged residue, is tolerated in position 1 of the peptide chain and the greatly decreased stability (half-life = 1 day) of des-Ala1-ferredoxin confirms the importance of the NH2-terminal residue for the stability of the protein. The relative stabilities of derivatives containing Ala1, but including a replacement for the normal Tyr2, are as follows: Native greater than [Trp2]- greater than or equal to [Phe2]- greater than [His2]- greater than [Leu2]- greater than [Pro2]ferredoxin. [Gly2]- and des-Ala1-Tyr2-apoferredoxin did not form stable derivatives upon reconstitution with iron and sulfide, nor did [3-NO2-Tyr2, 30]- and [Leu2,3-NO2-Tyr30]apoferredoxins. Other relatively stable and fully active derivatives prepared included: [3-NH2-Tyr30]-, [3-F-Phe2]-, and [2-F-Phe2]ferredoxin. The behavior of these various derivatives demonstrates the importance of the peptide chain for the stability of C. acidi-urici ferredoxin and shows that the activity of ferredoxin can be altered by a single amino acid substitution in the peptide chain.

The nucleotide sequence of the gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium acidiurici ("Clostridium acidi-urici") was determined. The synthetase mRNA initiation and termination regions were determined by primer extension and S1 nuclease mapping. Two potential -10 and -35 promoter regions were identified upstream of mRNA initiation. The terminator region was found to be in a large region of dyad symmetry. A comparison of the amino acid sequences of the monofunctional synthetase and the eucaryotic trifunctional enzyme, C1-tetrahydrofolate synthase, from Saccharomyces cerevisiae demonstrated a region of strong homology.

Antiserum to yeast C1-tetrahydrofolate (C1-H4folate) synthase reacts with other eukaryotic C1-H4folate synthases and prokaryotic 10-formyltetrahydrofolate (10-CHO-H4folate) synthetases [formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3] even though these enzymes vary in subunit size and function and probably vary widely in sequence. The comigration of the purified enzymes with the immunoreactive material establishes the specificity of the reaction for C1-H4folate synthase proteins. Reciprocal crossreaction of the antibody to Clostridium acidiurici 10-CHO-H4folate synthetase with the eukaryotic proteins indicates that such broad cross-species reactions are not specific to the antisera elicited in response to the yeast C1-H4folate synthase. These specific crossreactions among divergent species have been observed only on an electrophoretic transfer blot of a denaturing polyacrylamide gel. These observations may have been possible because of the sensitivity and specificity of the technique, which differ from more conventional immunochemical methods.

The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome.

Phosphorothioate analogues of ATP and isomers of CrATP and CrADP were used to examine the nucleotide stereoselectivity of formyltetrahydrofolate synthetase from procaryotic and eucaryotic sources. Substrate activity of the thio-ATP analogues increased as the site of sulfur substitution was changed from the gamma to the alpha position. Thus, adenine nucleotide analogues substituted with sulfur at an alpha nonbridging position (ATP alpha S isomers) were the most active, and ATP gamma S was inactive. When Mg2+ was used as the divalent cation, both enzymes showed a clear preference (higher V/Km value) for the Sp isomer of ATP beta S although the magnitude of the preference was greater with the bacterial enzyme. With Cd2+ as the divalent cation the Rp isomer was preferred, but the difference was greater with the yeast enzyme. Both (Sp)-MgATP beta S and (Rp)-CdATP beta S have the delta or right-hand screw sense configuration of the metal chelate ring. The reversal of stereoselectivity when the cation was changed indicates that the metal ion is coordinated to the beta-phosphate group. No stereoselectivity was observed when ATP alpha S isomers were used in the presence of Mg2+ or Cd2+, suggesting that the metals are not coordinated to the alpha-phosphate. ATP beta S was also found to be a competitive inhibitor of MgATP and CdATP, and the lowest Ki values were obtained with the lambda screw sense isomers. The screw sense isomers of bidentate CrATP exhibited no detectable substrate activity but were competitive inhibitors of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Clostridium pasteurianum genomic DNA indicates that the ferredoxin (Fd) gene is present in a single copy. The cloned Fd gene previously described (Graves, M.C., Mullenbach, G. T., and Rabinowitz, J. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1653-1657) was used to map in vivo and in vitro synthesized Fd transcripts. The in vivo mRNA was sized in two ways: by Northern hybridization analysis, and more directly from the known DNA sequence after the 5'- and 3'-termini were identified. The 5'-end was determined by primer extension-dideoxy sequencing and the 3'-end by S1 nuclease mapping. The monocistronic Fd mRNA contains about 255 nucleotides and, thus, is one of the shortest bacterial mRNAs yet described. We also examined the Fd transcripts produced by Escherichia coli transformed with the plasmid containing the Fd gene. E. coli RNA polymerase most likely recognizes the same promoter (P1) as the clostridial polymerase, and furthermore, efficiently uses an additional promoter (P2) that is poorly recognized by the normal host enzyme. For comparison, in vitro transcripts were generated by E. coli and Bacillus subtilis RNA polymerases. In vitro, only promoter P1 is used by either E. coli or B. subtilis RNA polymerase. The 3'-end of each of the four types of transcripts occurs essentially at the same location and maps to within a large dyad symmetry element. Comparison of the Fd promoter with other Gram-positive promoters reveals that some sequences outside of the traditional Pribnow and -35 regions are conserved. This analysis indicates that an "extended" promoter recognition site may be required in these organisms.

The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes.

13C NMR spectra of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins show that some 13C resonances of the aliphatic amino acid residues are shifted significantly from their corresponding resonance positions in the spectra of model polypeptides or apoferredoxin. Thirteen 13C resonances are shifted into the 80- to 120-ppm (from CS2) region, and have been assigned to the cysteinyl alpha and beta carbon atoms. The remaining shifted resonances in the 120- to 190-ppm region are tentatively assigned to amino acid residues that may be close to [4Fe-4S] clusters of the oxidized and reduced ferredoxins. The similarity in the shift pattern of the corresponding 13C resonances of the cysteinyl alpha and beta carbon atoms in the three ferredoxins studied suggests that the three-dimensional amino acid environments of the corresponding [4Fe-4S] clusters in each protein are similar.

We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin.

The (13)C-resonances of the 2',6'-ring carbon atoms of both tyrosyl residues of Clostridium acidi-urici ferredoxin are shifted downfield in the oxidized and reduced protein relative to these resonance positions in free tyrosine. These results show that both tyrosyl residues in the oxidized and reduced protein are in magnetically equivalent environments, and suggest that both tyrosyl residues are close to the two iron-sulfur clusters in the reduced and oxidized proteins and that each cluster is equally accessible to one reducing electron.