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X-ray Structure of Pyruvate Formate-Lyase in Complex with Pyruvate and CoA

HOW THE ENZYME USES THE CYS-418 THIYL RADICAL FOR PYRUVATE CLEAVAGE*
  • Andreas Becker
    Correspondence
    Supported by the Peter and Traudl Engelhorn Stiftung (Germany). To whom correspondence should be addressed. Tel.: 49-6221-486-276; Fax: 49-6221-486-437; E-mail:
    Affiliations
    From the Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Jahnstrasse 29, 69120 Heidelberg, Germany
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  • Wolfgang Kabsch
    Affiliations
    From the Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Jahnstrasse 29, 69120 Heidelberg, Germany
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The atomic coordinates and the structure factors (code 1h16, 1h17 and 1h18) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Open AccessPublished:August 05, 2002DOI:https://doi.org/10.1074/jbc.M205821200
      The glycyl radical enzyme pyruvate formate-lyase (PFL) synthesizes acetyl-CoA and formate from pyruvate and CoA. With the crystal structure of the non-radical form of PFL in complex with its two substrates, we have trapped the moment prior to pyruvate cleavage. The structure reveals how the active site aligns the scissile bond of pyruvate for radical attack, prevents non-radical side reactions of the pyruvate, and confines radical migration. The structure shows CoA in a syn conformation awaiting pyruvate cleavage. By changing to an anti conformation, without affecting the adenine binding mode of CoA, the thiol of CoA could pick up the acetyl group resulting from pyruvate cleavage.
      PFL
      pyruvate formate-lyase
      MOPS
      3-(N-morpholino)propanesulfonic acid
      oxa
      oxamate
      pyr
      pyruvate
      Proteins can domesticate amino acid radicals for enzymatic substrate conversions (Refs.
      • Reichard P.
      • Ehrenberg A.
      and
      • Knappe J.
      • Neugebauer F.A.
      • Blaschkowski H.P.
      • Gänzler M.
      , and for review, see Refs.
      • Stubbe J.
      • van der Donk W.A.
      ,
      • Eklund H.
      • Fontecave M.
      ,
      • Knappe J.
      • Wagner A.F.V.
      ). Among the best studied examples are ribonucleotide reductases and pyruvate formate-lyase (PFL).1 PFL and class III ribonucleotide reductase both depend on a glycyl radical (
      • Wagner A.F.V.
      • Frey M.
      • Neugebauer F.A.
      • Schäfer W.
      • Knappe J.
      ,
      • Sun X.
      • Ollagnier S.
      • Schmidt P.P.
      • Atta M.
      • Mulliez E.
      • Lepape L.
      • Eliasson R.
      • Graslund A.
      • Fontecave M.
      • Reichard P.
      • Sjoberg B.M.
      ) and display extensive structural and mechanistic similarities (
      • Logan D.T.
      • Andersson J.
      • Sjöberg B.-M.
      • Nordlund P.
      ,
      • Leppänen V.M.
      • Merckel M.C.
      • Ollis D.L.
      • Wong K.K.
      • Kozarich J.W.
      • Goldman A.
      ,
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Wagner A.F.V.
      ), which suggests that these enzymes derive from ancestors dated in the anaerobic world before the first appearance of DNA (
      • Reichard P.
      ).
      Active PFL, a homodimer (2 × 759 residues), is generated from the non-radical form by the removal of the pro-S Cα hydrogen of Gly-734, which is catalyzed by PFL-activase (
      • Frey M.
      • Rothe M.
      • Wagner A.F.V.
      • Knappe J.
      ). PFL catalyzes the reversible conversion of pyruvate and CoA into acetyl-CoA and formate, which has a central role in anaerobic glucose fermentation by Escherichia coli and other bacteria. The reaction divides into two half-reactions involving an acetyl-enzyme intermediate (E + pyruvate = acetyl-E + formate; acetyl-E + CoA = E + acetyl-CoA). The main participants in the reaction include Gly-734 for radical storage and a site carrying the acetyl intermediate consisting of two adjacent cysteinyl residues, Cys-418 and Cys-419. The glycyl radical is thought to generate a thiyl radical at Cys-418/419 needed for homolytic substrate cleavage.
      The recently reported (
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Wagner A.F.V.
      ) structure of PFL (non-radical form) in complex with oxamate, an isosteric and chemically inert analog of pyruvate, shows the close spatial proximity of the catalytic triad Gly-734, Cys-419, Cys-418 that could accommodate direct radical transfers from Gly-734 to Cys-418 via Cys-419. Oxamate fits into a compact pocket where its carboxamide C is juxtaposed with Cys-418 Sγ (3.3 Å). Based on the structure and biochemical information, along with theoretical data (
      • Himo F.
      • Eriksson L.A.
      ), a mechanism was proposed (
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Wagner A.F.V.
      ) that involves the following steps (Fig. 1 a). First half-reaction: pyruvate binding triggers generation of the Cys-418 thiyl radical by hydrogen transfer from Cys-418 S–H via Cys-419 to the glycyl radical; thiyl addition to the carbonyl C of pyruvate produces the tetrahedral oxyradical intermediate, which collapses into the acetyl thioester of Cys-418 and the formyl radical; the formyl radical is quenched to formate by the SH of Cys-419, thus producing the 419 thiyl. Second half-reaction: 419 thiyl generates the thiyl radical of CoA; the radical is replaced with the 418-linked acetyl resulting in acetyl-CoA.
      Figure thumbnail gr1a
      Figure 1Mechanism and structure of PFL.a, stages 1–7 of the proposed (
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Wagner A.F.V.
      ) catalytic mechanism sketch the first half-reaction, comprising radical transfer from Gly-734 via Cys-419 to Cys-418 with pyruvate (Pyr) bound in the active site, generation of the acetyl-enzyme intermediate, and release of formate (Fmt). Stages 8–10 depictS-acetyl transfer from the acetyl-enzyme to CoA and release of acetyl-CoA (AcCoA) during the second half-reaction. The radical is marked by a dot. b, stereoview of the PFL-dimer in complex with pyruvate and CoA. The structure mimicks theboxed stage 2 in the reaction cycle (a). The vicinal cysteines, Cα of Gly-734, CoA, and pyruvate are in ball and stick representation (green, sulfurs;gray, carbon; light blue, Gly-734 Cα).c, stereoview of the hypothetical structure of the acetyl-enzyme in complex with CoA in the anti conformation ready to pick up the acetyl group.
      Figure thumbnail gr1b
      Figure 1Mechanism and structure of PFL.a, stages 1–7 of the proposed (
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Wagner A.F.V.
      ) catalytic mechanism sketch the first half-reaction, comprising radical transfer from Gly-734 via Cys-419 to Cys-418 with pyruvate (Pyr) bound in the active site, generation of the acetyl-enzyme intermediate, and release of formate (Fmt). Stages 8–10 depictS-acetyl transfer from the acetyl-enzyme to CoA and release of acetyl-CoA (AcCoA) during the second half-reaction. The radical is marked by a dot. b, stereoview of the PFL-dimer in complex with pyruvate and CoA. The structure mimicks theboxed stage 2 in the reaction cycle (a). The vicinal cysteines, Cα of Gly-734, CoA, and pyruvate are in ball and stick representation (green, sulfurs;gray, carbon; light blue, Gly-734 Cα).c, stereoview of the hypothetical structure of the acetyl-enzyme in complex with CoA in the anti conformation ready to pick up the acetyl group.
      An earlier mechanistic proposal (
      • Knappe J.
      • Elbert S.
      • Frey M.
      • Wagner A.F.V.
      ) assumed the formation of a thiohemiketal between pyruvate and one of the active site cysteines. This possibility could not be ruled out by the available structure (
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Wagner A.F.V.
      ), as it contained the inert oxamate, which is principally unable to form a thiohemiketal-like compound. Moreover, the reported structures did not provide information on the binding site of the cosubstrate CoA/acetyl-CoA, since PFL shows no relationship to known folds of CoA binding proteins (
      • Engel C.
      • Wierenga R.
      ). We describe here the crystal structures of the non-radical form of PFL from E. coli in complex with pyruvate, with pyruvate and CoA, and for comparison, with oxamate and CoA. The new data support our view of the catalytic cycle (Fig.1 a) and show the binding site of the cosubstrate CoA for the first time.

      DISCUSSION

      An earlier proposal for the first half-reaction of the PFL mechanism (
      • Knappe J.
      • Elbert S.
      • Frey M.
      • Wagner A.F.V.
      ,
      • Knappe J.
      • Wagner A.F.V.
      ) assumed the initial formation of a thiohemiketal between pyruvate and the active site Cys-419. The reaction was thought to proceed by attack of the Cys-418 thiyl radical on the carboxylate C resulting in pyruvate cleavage. The formation of a thiohemiketal was inferred from gel filtration experiments at 4 °C which showed a [14C]pyruvate adduct to the non-radical form of PFL (
      • Knappe J.
      • Elbert S.
      • Frey M.
      • Wagner A.F.V.
      ). Recent structural work on PFL in complex with oxamate (
      • Becker A.
      • Fritz-Wolf K.
      • Kabsch W.
      • Knappe J.
      • Schultz S.
      • Wagner A.F.V.
      ) has led to a new view of the PFL mechanism (Fig. 1 a) that precludes thiohemiketal formation and assigns roles to the active site cysteines that differ from the earlier proposal. The new view was based on the assumption that pyruvate would display the same binding mode as the substrate analog oxamate. However, in contrast to pyruvate, oxamate is principally unable to form a thiohemiketal-like compound and for this reason the structural data were insufficient to rule out alternatives to the new view that might involve the formation of a thiohemiketal. The PFL+pyr and PFL+pyr+CoA structures described here are consistent with the new mechanistic proposal. The binding mode of pyruvate is very similar to that of oxamate. Moreover, pyruvate does not form a thiohemiketal, since the molecule does not display any significant deviations from planarity of its carbonyl group despite the close proximity of the carbonyl C to Cys-418 Sγ (Fig. 2 a). Apparently, pyruvate is tightly bound in the active site forming a kinetically stable complex with PFL, which could well explain the outcome of the gel filtration experiments (
      • Knappe J.
      • Elbert S.
      • Frey M.
      • Wagner A.F.V.
      ).
      The PFL+pyr and PFL+pyr+CoA structures indicate a weak bond between the C2 of pyruvate and the Sγ of Cys-418 thiol (bond length 2.6 Å), reminiscent of a launched nucleophilic attack on C2. In the radical form of PFL this attack would proceed upon hydrogen abstraction from Cys-418 thiol by Cys-419 thiyl. In the non-radical form the attack cannot proceed. The crystal structures are suggestive of a snapshot of a point on the reaction coordinate when the radical has moved from its storage location at Gly-734 to Cys-419, leaving Gly-734 Cα as a sp3 carbon (Fig. 1 a, boxed state). At this point the only difference between the radical and non-radical forms of PFL is the absence of a single hydrogen atom at Cys-419 Sγ in the enzyme. Thus, the PFL+pyr and PFL+pyr+CoA crystal structures provide excellent approximations to the corresponding radical enzyme-substrate complexes at the moment prior to generation of the Cys-418 thiyl. The observed structure of the active site, which is identical in the PFL+pyr and PFL+pyr+CoA crystals, is consistent with the radical mechanism of the first half reaction: 1) pyruvate is bound in the active site displaying its planar C2 (sp2) at sub-van der Waals distance to the Sγ of Cys-418 (Fig. 2). The active site is designed to fix pyruvate in a position that prevents the formation of a thiohemiketal (implying a tetrahedral sp3configuration at C2), which would render hydrogen abstraction from Cys-418 thiol impossible (
      • Knappe J.
      • Neugebauer F.A.
      • Blaschkowski H.P.
      • Gänzler M.
      ). The thiol of CoA in the PFL+pyr+CoA structure is located outside of the active site. Thus, besides the catalytic residues, there is no radical quenching group present in the active site that could interfere with pyruvate cleavage or lead to a loss of the radical.
      Consistent with biochemical data, our structures show that the presence of CoA is not required for pyruvate cleavage. If CoA is present during the first half-reaction, it binds in a waiting mode. Upon completion of the first half-reaction PFL must undergo some structural change that allows the thiol of CoA to reach the active site to pick up the acetyl from Cys-418. This requires a new binding mode for the ribose-pantetheine moiety of CoA that differs from that observed in the PFL+pyr+CoA structure.
      An approximate model for the new CoA binding mode could be obtained if one rotates the ribose-pantetheine moiety around theN-glycosidic bond. This could be visualized as a “fishing model” in which the angler (adenine) stands on a platform (Phe-149 and Asp-150) and moves the fishing rod (ribose-pantetheine moiety rotating from the syn to the anti conformation) through the air (solvent) into the water (protein) so that the hook (CoA thiol) catches the fish (acetyl at Cys-418). Indeed, as shown by model calculations, the ribose-pantetheine moiety can freely rotate through the solvent around the N-glycosidic bond from the unusual syn to the anti conformation so that the thiol reaches the active site (Figs. 1 c and 3 c). Since 5′-O-phosphorylated adenine nucleotides like CoA prefer the anti conformation in solution, thesyn/anti transition is favored by a gain in free energy. Presumably, the strictly conserved Arg-160 supports this transition by forming a new salt bridge with the pyrophosphate of CoA in the anti conformation. Moreover, the modeled new binding mode for CoA involves predominantly conserved residues and requires only moderate conformational changes of PFL that do not disrupt secondary structure. We expect that the new CoA binding site formed upon completion of the first half-reaction also allows for binding of a free CoA molecule (anti conformation) that approaches from solution, by-passing the waiting position seen in the crystal structure.
      The fixed binding site for the adenine of CoA throughout the reaction cycle proposed here is consistent with biochemical findings (

      Becker, A., Towards the Three-dimensional Structure of Pyruvate Formate-Lyase. Crystallization of the Protein and Search for Heavy Atom Derivatives-Photoaffinity Labeling Using S-ethyl-(8-azido)CoA.Ph.D. thesis, 1995, University of Heidelberg, Heidelberg, Germany.

      ) thatS-ethyl-(8-azido)-CoA and S-ethyl-(8-amino)-CoA, which prefer the syn conformation due to their bulky substituents at the C8 position, are competitive inhibitors (K I values 0.035 and 0.2 mm, respectively) of acetyl-CoA (K m = 0.05 mm (
      • Knappe J.
      • Blaschkowski H.P.
      • Gröbner P.
      • Schmitt T.
      )). This means that the CoA binding site in the radical form of PFL overlaps with that of the inhibitors.
      Taken together our structural data strongly support the proposed radical mechanism (Fig. 1 a) for the first half-cycle, provide clear evidence for the adenine binding site of CoA, and suggest how the thiol of CoA could reach the active site, formally replacing formate. It remains undecided by our data whether the presence of the acetyl at Cys-418, the release of formate, or the Cα configuration of Gly-734 (sp2) triggers formation of the new binding mode for the ribose-pantetheine moiety required for the second half-reaction. Our finding that CoA may adopt a waiting position ready to get involved after pyruvate cleavage is consistent with the indirect group transfer of PFL (ping-pong) mechanism. This could contribute to optimizing the enzyme for the synthesis of acetyl-CoA, a key compound in numerous biochemical pathways.

      Acknowledgments

      We are grateful to Joachim Knappe for stimulating discussions and providing facilities for PFL production. We thank A. F. Volker Wagner for providing E. coli strain 234M1 transformed with the expression vector p153E1, Klaus Scheffzek for help with the synchrotron measurements at the European Synchrotron Radiation Facility, the staff at the synchrotron facilities at the European Synchrotron Radiation Facility and Deutsches Elektronen Synchrotron, Hans Wagner for maintenance of the x-ray facilities at the MPI Heidelberg, John Wray for reading the manuscript, and Kenneth C. Holmes for continuous support.

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