YfiD of Escherichia coli and Y06I of Bacteriophage T4 as Autonomous Glycyl Radical Cofactors Reconstituting the Catalytic Center of Oxygen-Fragmented Pyruvate Formate-Lyase

• Published in: Biochemical and biophysical research communications 2001-07, Vol.285 (2), p.456-462
• Main Authors: Wagner, A.F.Volker, Schultz, Sabine, Bomke, Jörg, Pils, Thomas, Lehmann, Wolf D., Knappe, Joachim
• Format: Article
• Language: English
• Subjects: Acetyltransferases - chemistry; Acetyltransferases - metabolism; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacteriophage T4 - metabolism; Escherichia coli; Escherichia coli - metabolism; Free Radicals; Glycine; Kinetics; Models, Chemical; Models, Molecular; Molecular Sequence Data; Molecular Weight; Oxidative Stress; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Phage T4; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Viral Proteins - chemistry; Viral Proteins - metabolism; Y06I protein; YfiD protein
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.5186

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N–Cα of Gly734. A recombinant protein comprising the core of PFL (Ser1–Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase a heterooligomeric PFL enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a “spare part” for PFL's glycyl radical domain, utilized for rapid recovery of PFL activity (and thus ATP generation) in cells that have experienced oxidative stress.