YfiD of Escherichia coli and Y061 of bacteriophage T4 as autonomous glycylradical cofactors reconstituting the catalytic center of oxygen-fragmentedpyruvate formate-lyase

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (Pn) l eads to cleavage of the polypeptide backbone between N-C alpha of Gly734. A recombinant protein comprising the core of Pn (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewis e with the homologous T4 encoded Y06I protein, yielding upon reaction with Pn activase a heterooligomeric Pn enzyme that has full catalytic activity ( 35 U/nmol). Treatment of the activated complexes with oxygen led to cleavag e 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 (Y06 I). For the isolated fragments from Y06I, mass spectrometric analysis (nano ESI-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 th at YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for Pn's glycyl radical domain, utilized for rapid recovery of PFL activity land thus ATP generation) in cells that have experienced ox idative stress. (C) 2001 Academic Press.