Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase

Pyruvate formate-lyase (PFL) from Escherichia coli uses a radical mechanism to reversibly cleave the C1-C2 bond of pyruvate using the Gly 734 radical and two cysteine residues (Cys 418, Cys 419). We have determined by X-ray c rystallography the structures of PFL (non-radical form), its complex with t he substrate analog oxamate; and the C418A,C419A double mutant. The atomic model (a dimer of 759-residue monomers) comprises a 10-stranded beta/alpha barrel assembled in an antiparallel manner from two parallel five-stranded beta-sheets; this architecture resembles that of ribonucleotide reductases. Cry 734 and Cys 419, positioned at the tips of opposing hairpin loops, mee t in the apolar barrel center (C alpha-S gamma = 3.7 Angstrom). Oxamate fit s into a compact pocket where CZ is juxtaposed with Cys 418S gamma (3.3 Ang strom), which in turn is close to Cys 419S gamma (3.7 Angstrom). Our model of the active site is suggestive of a snapshot of the catalytic cycle, when the pyruvate-carbonyl awaits attack by the Cys 418 thiyl radical. We propo se a homolytic radical mechanism for PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions.