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 crystallography the structures of PFL (non-radical form), its complex with the substrate analog oxamate, and the C418A,C419A double mutant. The atomic model (a dimer of 759-residue monomers) comprises a 10-stranded β/α barrel assembled in an antiparallel manner from two parallel five-stranded β-sheets; this architecture resembles that of ribonucleotide reductases. Gly 734 and Cys 419, positioned at the tips of opposing hairpin loops, meet in the apolar barrel center (Cα–Sγ = 3.7 Å). Oxamate fits into a compact pocket where C2 is juxtaposed with Cys 418Sγ (3.3 Å), which in turn is close to Cys 419Sγ (3.7 Å). 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 propose a homolytic radical mechanismfor PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions.

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