PMID:10504733
| Citation |
Becker, A, Fritz-Wolf, K, Kabsch, W, Knappe, J, Schultz, S and Volker Wagner, AF (1999) Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. Nat. Struct. Biol. 6:969-75 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1038/13341 |
| Keywords |
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 |
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