Publications

• The free radical in pyruvate formate-lyase is located on glycine-734.

  Wagner A.F.V., Frey M., Neugebauer F.A., Schaefer W., Knappe J.

  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-labe ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 89:996-1000(1992) [PubMed] [EuropePMC]

• Radical mechanisms of enzymatic catalysis.

  Frey P.A.

  Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5'-deoxyadenosyl radical are discussed. In the first class, the mechanism of the interconversion of L-lysine and L-beta-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of whic ... >> More

  Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5'-deoxyadenosyl radical are discussed. In the first class, the mechanism of the interconversion of L-lysine and L-beta-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of which have been spectroscopically characterized. The reversible formation of the 5'-deoxyadenosyl radical takes place by the chemical cleavage of S-adenosylmethionine (SAM) reacting with the [4Fe-4S]+ center in LAM. In other reactions of SAM with iron-sulfur proteins, SAM is irreversibly consumed to generate the 5'-deoxyadenosyl radical, which activates an enzyme by abstracting a hydrogen atom from an enzymatic glycyl residue to form a glycyl radical. The glycyl radical enzymes include pyruvate formate-lyase, anaerobic ribonucleotide reductase from Escherichia coli, and benzylsuccinate synthase. Biotin synthase and lipoate synthase are SAM-dependent [4Fe-4S] proteins that catalyze the insertion of sulfur into unactivated C-H bonds, which are cleaved by the 5'-deoxyadenosyl radical from SAM. In the second class of enzymatic mechanisms using free radicals, adenosylcobalamin-dependent reactions, the 5'-deoxyadenosyl radical arises from homolytic cleavage of the cobalt-carbon bond, and it initiates radical reactions by abstracting hydrogen atoms from substrates. Three examples are described of suicide inactivation through the formation of exceptionally stable free radicals at enzymatic active sites. << Less

  Annu Rev Biochem 70:121-148(2001) [PubMed] [EuropePMC]

• Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme.

  Vey J.L., Yang J., Li M., Broderick W.E., Broderick J.B., Drennan C.L.

  Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur c ... >> More

  Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G(734). Our structures provide fundamental insights into the interactions between the activase and the G(734) loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G(734) of pyruvate formate-lyase. << Less

  Proc. Natl. Acad. Sci. U.S.A. 105:16137-16141(2008) [PubMed] [EuropePMC]

• Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom. Studies of [2H]glycine-substituted enzyme and peptides homologous to the glycine 734 site.

  Frey M., Rothe M., Wagner A.F., Knappe J.

  The active form of pyruvate formate-lyase (PFL) from Escherichia coli contains a glycyl radical in position 734 of the polypeptide chain which is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase) using S-adenosylmethionine (AdoMet) and dihydroflavodoxin as co ... >> More

  The active form of pyruvate formate-lyase (PFL) from Escherichia coli contains a glycyl radical in position 734 of the polypeptide chain which is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase) using S-adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates (Wagner, A.F. V., Frey, M., Neugebauer, F.A., Schäfer, W., and Knappe, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 996-1000). Studying radical synthesis with [2-2H]glycine-labeled PFL, we have now found stoichiometric incorporation of a 2H atom into the 5'-deoxyadenosine (dAdo) co-product via mass and NMR spectroscopic analyses. Furthermore, a series of peptides homologous to the Gly-734 site of PFL have been synthesized for analyzing recognition determinants of PFL activase. Peptides that proved active as substrates (monitored by [14C]dAdo formation from [14C]AdoMet) were also competitive inhibitors of PFL conversion to the radical form. In the sequence of the standard peptide Arg-Val-Ser-Gly-Tyr-Ala-Val, which corresponds to amino acid residues 731-737 of PFL, the Gly residue was replaceable by D-Ala (actually displaying enhanced efficiency), whereas a normal Ala totally abolished the interaction with PFL activase. Our results show that the radical in pyruvate formatelyase is produced by stereospecific abstraction of the pro-S hydrogen of glycine 734 by the 5'-dAdo radical generated in the active center of PFL activase. Gly-734 is probably located in a beta-turn segment of the protein. << Less

  J Biol Chem 269:12432-12437(1994) [PubMed] [EuropePMC]