The Autonomous Glycyl Radical Protein GrcA Restores Activity to Inactive Full-Length Pyruvate Formate-Lyase In Vivo

During glucose fermentation, Escherichia coli and many other microorganisms employ the glycyl radical enzyme (GRE) pyruvate formate-lyase (PflB) to catalyze the coenzyme A-dependent cleavage of pyruvate to formate and acetyl-coenzyme A (CoA). Due to its extreme reactivity, the radical in PflB must be controlled carefully and, once generated, is particularly susceptible to dioxygen. Exposure to oxygen of the radical on glycine residue 734 of PflB results in cleavage of the polypeptide chain and consequent inactivation of the enzyme. Two decades ago, a small 14-kDa protein called YfiD (now called autonomous glycyl radical cofactor [GrcA]) was shown to be capable of restoring activity to O₂-inactivated PflB in vitro; however, GrcA has never been shown to have this function in vivo. By constructing a strain with a chromosomally encoded PflB enzyme variant with a G734A residue exchange, we could show that cells retained near-wild type fermentative growth, as well as formate and H₂ production; H₂ is derived by enzymatic disproportionation of formate. Introducing a grcA deletion mutation into this strain completely prevented formate and H₂ generation and reduced anaerobic growth. We could show that the conserved glycine at position 102 on GrcA was necessary for GrcA to restore PflB activity and that this recovered activity depended on the essential cysteine residues 418 and 419 in the active site of PflB. Together, our findings demonstrate that GrcA is capable of restoring in vivo activity to inactive full-length PflB and support a model whereby GrcA displaces the C-terminal glycyl radical domain to rescue the catalytic function of PflB. IMPORTANCE Many facultative anaerobic microorganisms use glycyl radical enzymes (GREs) to catalyze chemically challenging reactions under anaerobic conditions. Pyruvate formate-lyase (PflB) is a GRE that catalyzes cleavage of the carbon-carbon bond of pyruvate during glucose fermentation. The problem is that glycyl radicals are destroyed readily, especially by oxygen. To protect and restore activity to inactivated PflB, bacteria like Escherichia coli have a small autonomous glycyl radical cofactor protein called GrcA, which functions to rescue inactivated PflB. To date, this proposed function of GrcA has only been demonstrated in vitro. Our data reveal that GrcA rescues and restores enzyme activity to an inactive full-length form of PflB in vivo. These results have important implications for the evolution of radical-based enzyme mechanisms.