The free radical in pyruvate formate-lyase is located on glycine-734 (original) (raw)

Abstract

Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.

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  1. CLARK A. J. Genetic analysis of a "double male" strain of Escherichia coli K-12. Genetics. 1963 Jan;48:105–120. doi: 10.1093/genetics/48.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [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. Debus R. J., Barry B. A., Babcock G. T., McIntosh L. Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci U S A. 1988 Jan;85(2):427–430. doi: 10.1073/pnas.85.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dixon H. B. The conversion of glyoxyloyl groups into glycyl groups and their formation from maleyl residues. Biochem J. 1968 Mar;107(1):124–126. doi: 10.1042/bj1070124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eliasson R., Fontecave M., Jörnvall H., Krook M., Pontis E., Reichard P. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor. Proc Natl Acad Sci U S A. 1990 May;87(9):3314–3318. doi: 10.1073/pnas.87.9.3314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gautney L. L., Jr, Miyagawa I. ESR study of hydrogen exchange reaction in x-ray-irradiated glycine. Radiat Res. 1975 Apr;62(1):12–17. [PubMed] [Google Scholar]
  8. Karthein R., Dietz R., Nastainczyk W., Ruf H. H. Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction. Eur J Biochem. 1988 Jan 15;171(1-2):313–320. doi: 10.1111/j.1432-1033.1988.tb13792.x. [DOI] [PubMed] [Google Scholar]
  9. Kirino Y., Taniguchi H. An ESR study of the acid dissociation of NH protons. 1. Linear peptide radicals and related radicals. J Am Chem Soc. 1976 Aug 18;98(17):5089–5096. doi: 10.1021/ja00433a007. [DOI] [PubMed] [Google Scholar]
  10. Knappe J., Neugebauer F. A., Blaschkowski H. P., Gänzler M. Post-translational activation introduces a free radical into pyruvate formate-lyase. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1332–1335. doi: 10.1073/pnas.81.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Moss M. L., Frey P. A. Activation of lysine 2,3-aminomutase by S-adenosylmethionine. J Biol Chem. 1990 Oct 25;265(30):18112–18115. [PubMed] [Google Scholar]
  14. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nordlund P., Sjöberg B. M., Eklund H. Three-dimensional structure of the free radical protein of ribonucleotide reductase. Nature. 1990 Jun 14;345(6276):593–598. doi: 10.1038/345593a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Reichard P., Ehrenberg A. Ribonucleotide reductase--a radical enzyme. Science. 1983 Aug 5;221(4610):514–519. doi: 10.1126/science.6306767. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Sawers G., Wagner A. F., Böck A. Transcription initiation at multiple promoters of the pfl gene by E sigma 70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo. J Bacteriol. 1989 Sep;171(9):4930–4937. doi: 10.1128/jb.171.9.4930-4937.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  21. Sivaraja M., Goodin D. B., Smith M., Hoffman B. M. Identification by ENDOR of Trp191 as the free-radical site in cytochrome c peroxidase compound ES. Science. 1989 Aug 18;245(4919):738–740. doi: 10.1126/science.2549632. [DOI] [PubMed] [Google Scholar]
  22. Stubbe J. Ribonucleotide reductases: amazing and confusing. J Biol Chem. 1990 Apr 5;265(10):5329–5332. [PubMed] [Google Scholar]
  23. Taniguchi H., Kirino Y. An ESR study of the acid dissociation of NH protons. 2. Cyclic peptide radicals and related radicals. J Am Chem Soc. 1977 May 25;99(11):3625–3631. doi: 10.1021/ja00453a019. [DOI] [PubMed] [Google Scholar]
  24. Taylor A. L., Trotter C. D. Revised linkage map of Escherichia coli. Bacteriol Rev. 1967 Dec;31(4):332–353. doi: 10.1128/br.31.4.332-353.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Whittaker M. M., Whittaker J. W. A tyrosine-derived free radical in apogalactose oxidase. J Biol Chem. 1990 Jun 15;265(17):9610–9613. [PubMed] [Google Scholar]