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

Nature Structural Biology volume 6, pages 969–975 (1999)Cite this article

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 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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Figure 1: Evidence for the binding of oxamate in the active site.
Figure 2: Stereo view of Cα traces of the PFL dimer in complex with oxamate.
Figure 3: Schematic representation of secondary structure of a PFL monomer.
Figure 4: Ribbon diagram of a monomer in stereo.
Figure 5: Stereo view of the (2Fo - Fc) electron density map, contoured at 1.05σ,
Figure 6: Structural similarity between PFL and NrdD.
Figure 7: Structure of the active site.
Figure 8: Proposed catalytic mechanism of PFL.

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Acknowledgements

We thank D. Logan for providing class III ribonucleotide reductase coordinates preceding their general release, G. Sawers for the host E. coli strain RM221, D. Madden, K. Scheffzek and I. Schlichting for critical discussions and help, C. Lantwin and E. Pai for contributions in the early phase of the project, H. Wagner for maintenance of the X-ray facilities at the MPI Heidelberg, and K. Holmes for continuous support. Financial support of this work (by grants to J.K.) came from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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  1. Abteilung Biophysik, Max-Planck-Institut für medizinische Forschung, Jahnstrabe 29, Heidelberg, 69120, Germany

    Andreas Becker, Karin Fritz-Wolf & Wolfgang Kabsch

  2. Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universität, Im Neuenheimer Feld 501, Heidelberg, , 69120, Germany

    Joachim Knappe, Sabine Schultz & A. F. Volker Wagner

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  1. Andreas Becker
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Correspondence to Wolfgang Kabsch or Joachim Knappe.

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Becker, A., Fritz-Wolf, K., Kabsch, W. et al. Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase . Nat Struct Mol Biol 6, 969–975 (1999). https://doi.org/10.1038/13341

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