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Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase

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Abstract

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (μmol·min-1·mg-1) of FTHFS in cell free extracts of CRL47 were 28–89 when assayed at 50°C and pH8. This was from 3–10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.

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Abbreviations

FTHFS:

formyltetrahydrofolate synthetase

kb:

kilobase

H4-folate:

tetrahydrofolate

SDS:

sodium dodecyl sulfate

References

  • Dev IK, Harvey RJ (1978) A complex of N5N10-methylenetetrahydrofolate dehydrogenase and N5N10-methenyltetrahydrofolate cyclohydrolase in Escherichia coli. Purification, subunit structure, and allosteric inhibitions by N10-formyltetrahydrofolate. J Biol Chem 259:6085–6089

    Google Scholar 

  • Elliott JI, Ljungdahl LG (1982) Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum. Arch Biochem Biophys 215:245–252

    Google Scholar 

  • Fuchs G (1986) CO2 fixation in acetogenic bacteria: variations on a theme. FEMS Microbiol Rev 39:181–213

    Google Scholar 

  • Harmony JAK, Himes RH (1975) The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits. J Biol Chem 250:8049–8054

    Google Scholar 

  • Himes RH, Harmony JAK (1973) Formyltetrahydrofolate synthetase CRC Crit Rev Biochem 1:501–535

    Google Scholar 

  • Johnson DA, Gautsch JW, Sportsman JR, Elder JH (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose Gene Anal Techn 1:3–8

    Google Scholar 

  • Kushner SR (1978) An improved method for transformation of Escherichia coli with Col El derived plasmids. In: Boyer HB, Nicosia S (eds) Genetic engineering, Elsevier/North-Holland Biomedical Press, Amsterdam, pp 17–23

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685

    Google Scholar 

  • Ljungdahl LG (1979) Physiology of thermophilic bacteria. Adv Microbial Physiol 19:149–243

    Google Scholar 

  • Ljungdahl LG (1984) Other functions of folates. In: Blakley RL, Benkovic SJ (eds) Folates and pterin, vol 1. Wiley-Interscience, New York, pp 555–579

    Google Scholar 

  • Ljungdahl LG (1986) The autotrophic pathway of acetate synthesis in acetogenic bacteria. Ann Rev Microbiol 40:415–450

    Google Scholar 

  • Ljungdahl LG, Andreesen JR (1978) Formate dehydrogenase, a selenium-tungsten enzyme from Clostridium thermoaceticum. Meth Enzymol 53:360–372

    Google Scholar 

  • Ljungdahl LG, Brewer JM, Neece SH, Fairwell T (1970) Purification, stability, and composition of the formyltetrahydrofolate synthetase from Clostridium thermoaceticum. J Biol Chem 245:4791–4797

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent J Biol Chem 193:265–275

    Google Scholar 

  • MacKenzie RE (1984) Biogenesis and interconversion of substituted tetrahydrofolates. In: Blakley RL, Benkovic SJ (eds) Folates and pterins, vol 1. Wiley-Interscience, New York, pp 255–306

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 1–545

    Google Scholar 

  • Marmur J (1961) A procedure for the isolation of desoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218

    Google Scholar 

  • Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base specific chemical cleavages. Meth Enzymol 65:499–559

    Google Scholar 

  • Mayer F, Elliott JI, Sherod D, Ljungdahl LG (1982) Formyltetrahydrofolate synthetase from Clostridium thermoaceticum. An electron microscopic study and specific interaction of the enzyme with ATP and ADP. Eur J Biochem 124:397–404

    Google Scholar 

  • O'Brien WE, Brewer JM, Ljungdahl LG (1976) Chemical, physical, and enzymatic comparisons of formyltetrahydrofolate synthetase from thermo- and mesophilic clostridia. In: Zuber H (ed) Enzymes and proteins from thermophilic microorganisms, structure and functions. Birkhäuser, Boston Basel Stuttgart, pp 249–262

    Google Scholar 

  • O'Brien WE, Ljungdahl LG (1972) Fermentation of fructose and synthesis of acetate from carbon dioxide by Clostridium formicoaceticum. J Bacteriol 109:626–632

    Google Scholar 

  • Prasher D, McCann RO, Cormier MJ (1985) Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. Biochem Biophys Res Commun 126:1259–1268

    Google Scholar 

  • Rabinowitz JC, Pricer WE Jr (1962) Formyltetrahydrofolate synthetase. I. Isolation and crystallization of the enzyme. J Biol Chem 237:2898–2902

    Google Scholar 

  • Salvesen G, Farley D, Shuman J, Przybyla A, Reilley C, Travis J (1987) Molecular cloning of human cathepsin G: Structural similarity to mast cell and cytotoxic T-lymphocyte proteinases. Biochemistry 26:2289–2293

    Google Scholar 

  • Schiefer-Ullrich H, Wagner R, Dürre P, Andreesen JR (1984) Comparative studies on physiology and taxonomy of obligately purinolytic clostridia. Arch Microbiol 138:345–353

    Google Scholar 

  • Shoaf WT, Neece SH, Ljungdahl LG (1974) Effects of temperature and ammonium ions on formyltetrahydrofolate synthetase from Clostridium thermoaceticum. Biochim Biophys Acta 334:448–458

    Google Scholar 

  • Southern F (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517

    Google Scholar 

  • Taub F, Thompson EB (1982) An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters. Anal Biochem 126:222–230

    Google Scholar 

  • Towbin H, Stachelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Google Scholar 

  • Waber LJ, Wood HG (1979) Mechanism of acetate synthesis from CO2 by Clostridium acidiurici. J Bacteriol 140:468–478

    Google Scholar 

  • Wahl GM, Stern M, Stark GR (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethalpaper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683–3687

    Google Scholar 

  • Whitehead TR, Rabinowitz JC (1986) Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici (“Clostridium acidiurici”). J Bacteriol 167:205–209

    Google Scholar 

  • Whiteley HR, Osborn MJ, Huennekens FM (1959) Purification and properties of the formate activating enzyme from Micrococcus aerogenes. J Biol Chem 234:1538–1543

    Google Scholar 

  • Wood HG, Ragsdale SW, Pezacka E (1986) The acetyl-CoA pathway of autotrophic growth. FEMS Microbiol Rev 39:345–362

    Google Scholar 

  • Wood WI, Gitschier J, Lasky LA, Lawn RM (1985) Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci USA 82:1585–1588

    Google Scholar 

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Author notes

  1. Charles R. Lovell

    Present address: Department of Biology, University of South Carolina, 29208, Columbia, SC, USA

Authors and Affiliations

  1. Department of Biochemistry, University of Georgia, 30602, Athens, GA, USA

    Charles R. Lovell, Alan Przybyla & Lars G. Ljungdahl

  2. Center for Biological Resource Recovery, University of Georgia, 30602, Athens, GA, USA

    Charles R. Lovell, Alan Przybyla & Lars G. Ljungdahl

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  1. Charles R. Lovell
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  2. Alan Przybyla
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  3. Lars G. Ljungdahl
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Lovell, C.R., Przybyla, A. & Ljungdahl, L.G. Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase. Arch Microbiol 149, 280–285 (1988). https://doi.org/10.1007/BF00411642

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  • DOI: https://doi.org/10.1007/BF00411642

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