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 - PubMed (original) (raw)
. 1994 Apr 29;269(17):12432-7.
Affiliations
- PMID: 8175649
Free article
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
M Frey et al. J Biol Chem. 1994.
Free article
Abstract
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.
Similar articles
- A dehydroalanyl residue can capture the 5'-deoxyadenosyl radical generated from S-adenosylmethionine by pyruvate formate-lyase-activating enzyme.
Wagner AF, Demand J, Schilling G, Pils T, Knappe J. Wagner AF, et al. Biochem Biophys Res Commun. 1999 Jan 19;254(2):306-10. doi: 10.1006/bbrc.1998.9931. Biochem Biophys Res Commun. 1999. PMID: 9918833 - Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form.
Külzer R, Pils T, Kappl R, Hüttermann J, Knappe J. Külzer R, et al. J Biol Chem. 1998 Feb 27;273(9):4897-903. doi: 10.1074/jbc.273.9.4897. J Biol Chem. 1998. PMID: 9478932 - Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme.
Crain AV, Broderick JB. Crain AV, et al. J Biol Chem. 2014 Feb 28;289(9):5723-9. doi: 10.1074/jbc.M113.496877. Epub 2013 Dec 12. J Biol Chem. 2014. PMID: 24338017 Free PMC article. - A glycyl radical solution: oxygen-dependent interconversion of pyruvate formate-lyase.
Sawers G, Watson G. Sawers G, et al. Mol Microbiol. 1998 Aug;29(4):945-54. doi: 10.1046/j.1365-2958.1998.00941.x. Mol Microbiol. 1998. PMID: 9767563 Review. - A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli.
Knappe J, Sawers G. Knappe J, et al. FEMS Microbiol Rev. 1990 Aug;6(4):383-98. doi: 10.1111/j.1574-6968.1990.tb04108.x. FEMS Microbiol Rev. 1990. PMID: 2248795 Review.
Cited by
- Mechanistic Studies of Radical SAM Enzymes: Pyruvate Formate-Lyase Activating Enzyme and Lysine 2,3-Aminomutase Case Studies.
Byer AS, McDaniel EC, Impano S, Broderick WE, Broderick JB. Byer AS, et al. Methods Enzymol. 2018;606:269-318. doi: 10.1016/bs.mie.2018.04.013. Epub 2018 Jul 7. Methods Enzymol. 2018. PMID: 30097096 Free PMC article. - Biochemical and physiological characterization of the pyruvate formate-lyase Pfl1 of Chlamydomonas reinhardtii, a typically bacterial enzyme in a eukaryotic alga.
Hemschemeier A, Jacobs J, Happe T. Hemschemeier A, et al. Eukaryot Cell. 2008 Mar;7(3):518-26. doi: 10.1128/EC.00368-07. Epub 2008 Feb 1. Eukaryot Cell. 2008. PMID: 18245276 Free PMC article. - Mechanisms for control of biological electron transfer reactions.
Williamson HR, Dow BA, Davidson VL. Williamson HR, et al. Bioorg Chem. 2014 Dec;57:213-221. doi: 10.1016/j.bioorg.2014.06.006. Epub 2014 Jul 12. Bioorg Chem. 2014. PMID: 25085775 Free PMC article. Review. - Identification of a unique Fe-S cluster binding site in a glycyl-radical type microcompartment shell protein.
Thompson MC, Wheatley NM, Jorda J, Sawaya MR, Gidaniyan SD, Ahmed H, Yang Z, McCarty KN, Whitelegge JP, Yeates TO. Thompson MC, et al. J Mol Biol. 2014 Sep 23;426(19):3287-3304. doi: 10.1016/j.jmb.2014.07.018. Epub 2014 Aug 4. J Mol Biol. 2014. PMID: 25102080 Free PMC article. - Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase.
Arnau J, Jørgensen F, Madsen SM, Vrang A, Israelsen H. Arnau J, et al. J Bacteriol. 1997 Sep;179(18):5884-91. doi: 10.1128/jb.179.18.5884-5891.1997. J Bacteriol. 1997. PMID: 9294449 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Miscellaneous