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. 1996 Apr;178(8):2440–2444. doi: 10.1128/jb.178.8.2440-2444.1996

Molecular characterization of the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum.

G Weidner 1, G Sawers 1
PMCID: PMC177960  PMID: 8636053

Abstract

Formate is the major source of C1 units in many species of the genus Clostridium. In this study we have cloned and characterized the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum. The genetic and transcriptional organizations of the genes and the high level of homology exhibited by the respective gene products to their Escherichia coli counterparts indicate strong evolutionary conservation of these enzymes.

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Selected References

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  1. Balch W. E., Wolfe R. S. New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressureized atmosphere. Appl Environ Microbiol. 1976 Dec;32(6):781–791. doi: 10.1128/aem.32.6.781-791.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. Conradt H., Hohmann-Berger M., Hohmann H. P., Blaschkowski H. P., Knappe J. Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties. Arch Biochem Biophys. 1984 Jan;228(1):133–142. doi: 10.1016/0003-9861(84)90054-7. [DOI] [PubMed] [Google Scholar]
  4. Ike Y., Craig R. A., White B. A., Yagi Y., Clewell D. B. Modification of Streptococcus faecalis sex pheromones after acquisition of plasmid DNA. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5369–5373. doi: 10.1073/pnas.80.17.5369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jungermann K., Kirchniawy H., Thauer R. K. Ferredoxin dependent CO-2 reduction to formate in Clostridium pasteurianum. Biochem Biophys Res Commun. 1970 Nov 9;41(3):682–689. doi: 10.1016/0006-291x(70)90067-7. [DOI] [PubMed] [Google Scholar]
  6. Kessler D., Herth W., Knappe J. Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli. J Biol Chem. 1992 Sep 5;267(25):18073–18079. [PubMed] [Google Scholar]
  7. Knappe J., Blaschkowski H. P., Gröbner P., Schmitt T. Pyruvate formate-lyase of Escherichia coli: the acetyl-enzyme intermediate. Eur J Biochem. 1974 Dec 16;50(1):253–263. doi: 10.1111/j.1432-1033.1974.tb03894.x. [DOI] [PubMed] [Google Scholar]
  8. Knappe J., Sawers G. A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli. FEMS Microbiol Rev. 1990 Aug;6(4):383–398. doi: 10.1111/j.1574-6968.1990.tb04108.x. [DOI] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lindmark D. G., Paolella P., Wood N. P. The pyruvate formate-lyase system of Streptococcus faecalis. I. Purification and properties of the formate-pyruvate exchange enzyme. J Biol Chem. 1969 Jul 10;244(13):3605–3612. [PubMed] [Google Scholar]
  12. Lyons L. B., Zinder N. D. The genetic map of the filamentous bacteriophage f1. Virology. 1972 Jul;49(1):45–60. doi: 10.1016/s0042-6822(72)80006-0. [DOI] [PubMed] [Google Scholar]
  13. O'Brien R. W., Morris J. G. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol. 1971 Nov;68(3):307–318. doi: 10.1099/00221287-68-3-307. [DOI] [PubMed] [Google Scholar]
  14. Plaga W., Frank R., Knappe J. Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate). Eur J Biochem. 1988 Dec 15;178(2):445–450. doi: 10.1111/j.1432-1033.1988.tb14468.x. [DOI] [PubMed] [Google Scholar]
  15. Rödel W., Plaga W., Frank R., Knappe J. Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences. Eur J Biochem. 1988 Oct 15;177(1):153–158. doi: 10.1111/j.1432-1033.1988.tb14356.x. [DOI] [PubMed] [Google Scholar]
  16. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sauter M., Sawers R. G. Transcriptional analysis of the gene encoding pyruvate formate-lyase-activating enzyme of Escherichia coli. Mol Microbiol. 1990 Mar;4(3):355–363. doi: 10.1111/j.1365-2958.1990.tb00603.x. [DOI] [PubMed] [Google Scholar]
  18. Sawers G., Böck A. Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12. J Bacteriol. 1988 Nov;170(11):5330–5336. doi: 10.1128/jb.170.11.5330-5336.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sawers G., Böck A. Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression. J Bacteriol. 1989 May;171(5):2485–2498. doi: 10.1128/jb.171.5.2485-2498.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sawers G. Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR. Mol Microbiol. 1993 Nov;10(4):737–747. doi: 10.1111/j.1365-2958.1993.tb00944.x. [DOI] [PubMed] [Google Scholar]
  21. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  23. Suppmann B., Sawers G. Isolation and characterization of hypophosphite--resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter. Mol Microbiol. 1994 Mar;11(5):965–982. doi: 10.1111/j.1365-2958.1994.tb00375.x. [DOI] [PubMed] [Google Scholar]
  24. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Takahashi S., Abbe K., Yamada T. Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties. J Bacteriol. 1982 Mar;149(3):1034–1040. doi: 10.1128/jb.149.3.1034-1040.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thauer R. K., Kirchniawy F. H., Jungermann K. A. Properties and function of the pyruvate-formate-lyase reaction in clostridiae. Eur J Biochem. 1972 May 23;27(2):282–290. doi: 10.1111/j.1432-1033.1972.tb01837.x. [DOI] [PubMed] [Google Scholar]
  27. Thauer R. K., Rupprecht E., Jungermann K. The synthesis of one-carbon units from CO(2) via a new ferredoxin dependent monocarboxylic acid cycle. FEBS Lett. 1970 Jun 27;8(5):304–307. doi: 10.1016/0014-5793(70)80293-9. [DOI] [PubMed] [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Unkrig V., Neugebauer F. A., Knappe J. The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite. Eur J Biochem. 1989 Oct 1;184(3):723–728. doi: 10.1111/j.1432-1033.1989.tb15072.x. [DOI] [PubMed] [Google Scholar]
  30. Wagner A. F., Frey M., Neugebauer F. A., Schäfer W., Knappe J. The free radical in pyruvate formate-lyase is located on glycine-734. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):996–1000. doi: 10.1073/pnas.89.3.996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wood N. P., Jungermann K. Inactivation of the pyruvate formate lyase of Clostridium butyricum. FEBS Lett. 1972 Oct 15;27(1):49–52. doi: 10.1016/0014-5793(72)80407-1. [DOI] [PubMed] [Google Scholar]
  32. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  33. Zinoni F., Birkmann A., Stadtman T. C., Böck A. Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4650–4654. doi: 10.1073/pnas.83.13.4650. [DOI] [PMC free article] [PubMed] [Google Scholar]

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