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

Nat Struct Biol. 1999 Oct;6(10):969-75. doi: 10.1038/13341.

Abstract

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 beta/alpha barrel assembled in an antiparallel manner from two parallel five-stranded beta-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 (Calpha-Sgamma = 3.7 A). Oxamate fits into a compact pocket where C2 is juxtaposed with Cys 418Sgamma (3.3 A), which in turn is close to Cys 419Sgamma (3.7 A). 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 mechanism for PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyltransferases / chemistry*
  • Acetyltransferases / genetics
  • Acetyltransferases / metabolism*
  • Amino Acid Substitution
  • Binding Sites
  • Catalysis
  • Crystallization
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism
  • Dimerization
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Glycine / chemistry
  • Glycine / genetics
  • Glycine / metabolism
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Oxamic Acid / chemistry
  • Oxamic Acid / metabolism
  • Protein Structure, Secondary
  • Pyruvic Acid / metabolism
  • Ribonucleotide Reductases / chemistry
  • Ribonucleotide Reductases / genetics
  • Structure-Activity Relationship

Substances

  • Pyruvic Acid
  • Ribonucleotide Reductases
  • Acetyltransferases
  • formate C-acetyltransferase
  • Cysteine
  • Oxamic Acid
  • Glycine

Associated data

  • PDB/1CM5
  • PDB/2PFL
  • PDB/3PFL