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 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.
Becker
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A
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Jahnstrabe 29, 69120 Heidelberg, Germany.
Fritz-Wolf
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K
Kabsch
W
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Knappe
J
J
Schultz
S
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Volker Wagner
A F
AF
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010072:Oxamic Acid / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D019289:Pyruvic Acid / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D019289:Pyruvic Acid; D012264:Ribonucleotide Reductases; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D003545:Cysteine; D010072:Oxamic Acid; D005998:Glycine