In vivo evidence for glycyl radical insertion into a catalytically inactive variant of pyruvate formate-lyase.

The dimeric glycyl radical enzyme pyruvate formate-lyase (PflB; formate acetyltransferase 1) cleaves pyruvate with hypothetical half-site reactivity to formate and acetyl-CoA. The radical introduced onto residue G734 of PflB is transiently transferred to C419 of an adjacent cysteine pair (C418/C419) during catalysis, but it is unclear whether glycyl radical formation is dependent on C419 in vivo. We show here that a deficiency in formate production of an Escherichia coli strain synthesizing a PflB variant, but lacking the autonomous glycyl radical cofactor, GrcA, could be restored by reintroducing plasmid-encoded native PflB, but not by a PflB variant, indicating that PflB cannot replace GrcA. Oxygen-dependent polypeptide cleavage of PflB indicated stable glycyl radical incorporation; however, these data did not support half-site reactivity. These in vivo findings demonstrate that glycyl radical formation is independent of subsequent radical transfer from G734 to C419, which occurs intramolecularly. Impact statement Active, dimeric pyruvate formate-lyase has a stable radical on a glycine residue, which transiently abstracts a H-atom from a cysteine, generating a catalytic thiyl radical. Glycyl radical generation is independent of glycine-to-cysteine radical-transfer in vivo. Radical-transfer is intramolecular and the enzyme does not appear to exhibit half-site reactivity.