YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.
Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N-Calpha 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.
Wagner
A F
AF
Biochemie-Zentrum Heidelberg (BZH), Ruprecht-Karls-Universität, Im Neuenheimer Feld 501, 69120 Heidelberg, Germany.
Schultz
S
S
Bomke
J
J
Pils
T
T
Lehmann
W D
WD
Knappe
J
J
D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005609:Free Radicals; D005998:Glycine; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D005609:Free Radicals; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D005998:Glycine