One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography

Abstract

Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (OOR) metabolizes oxalate, generating two molecules of CO2 and two low-potential electrons, thus providing both the carbon and reducing equivalents for operation of the Wood-Ljungdahl pathway of acetogenesis. Here we present structures of OOR in which two different reaction intermediate bound states have been trapped: the covalent adducts between TPP and oxalate and between TPP and CO2. These structures, along with the previously determined structure of substrate-free OOR, allow us to visualize how active site rearrangements can drive catalysis. Our results suggest that OOR operates via a bait-and-switch mechanism, attracting substrate into the active site through the presence of positively charged and polar residues, and then altering the electrostatic environment through loop and side chain movements to drive catalysis. This simple but elegant mechanism explains how oxalate, a molecule that humans and most animals cannot break down, can be used for growth by acetogenic bacteria.

Keywords: carbon dioxide; oxalate; oxidoreductase; thiamine pyrophosphate.

Fig. 1.

OOR and PFOR catalyze the oxidation of oxalate and pyruvate, respectively, reducing two ferredoxin equivalents in the process. (A) The reaction scheme for OOR. (B) The reaction scheme for PFOR.

Fig. 2.

Substrate-bound intermediates in OOR and corresponding composite-omit 2Fo-Fc (gray mesh) electron density maps contoured at 1 σ (see SI Appendix, Fig. S2 for stereoviews). (A) Composite-omit electron density of the COOM-TPP intermediate (white) and the side chains of nearby residues including Arg31α, Arg109α, and Asp116α (green) are shown as sticks. (B) Composite-omit electron density of the carboxy-TPP intermediate from monomer 1 of the oxalate cocrystal is shown with the same representation as in A, but, due to a conformational change of the Switch loop, Phe117α is now shown instead of Asp116α.

Fig. 3.

Snapshots of OOR catalysis compared with PFOR with close interactions, <4 Å, shown as dashed lines. (A) The native structure of OOR (PDB ID: 5C4I) revealed a substrate-binding pocket formed by hydrophilic and positively charged residues. Select active site residues are also shown as stick models. Carbon atoms from domain I are colored green, whereas those from domain VI are colored dark red. Oxygen atoms are colored red, nitrogen atoms are colored blue, sulfur atoms are colored yellow, and phosphorous atoms are colored dark orange. Active site water molecules are shown as red spheres. For distances, see SI Appendix, Fig. S4. Inset shows a model of oxalate (with carbons colored dark gray) docked into the substrate-binding pocket based on a structure of PFOR with pyruvate (19). Proposed interactions between oxalate and surrounding protein side chains and water molecules are indicated with dashed lines. (B) The COOM-TPP intermediate from monomer 2 of the oxalate-soaked crystal is shown as in A. (C) The carboxy-TPP intermediate from monomer 1 of the oxalate cocrystal is shown as in A. (D) The carboxy-TPP intermediate from monomer 2 of the oxalate cocrystal is shown as above. (E) The lactyl-TPP intermediate observed in Da PFOR (19) (PDB ID: 2C3P) is shown with a similar representation as A, but with all carbons colored white. Substrate-binding residues are shown as sticks. (F) The HE-TPP intermediate observed in Da PFOR (18) (PDB ID: 1KEK) is shown as in E.

Fig. 4.

Residues 113–117 of chain α in OOR form a Switch loop that has two alternate conformations. (A) The Asp-out conformation of the native OOR structure (PDB ID: 5C4I) is shown with the Switch residues as stick models. In this conformation, Phe117α is oriented toward the substrate-binding pocket. Arrows from Pro113α, Asp116α, and Phe117α indicate the direction of movement of these residues to go from Asp-out to Asp-in. (B) The Asp-in conformation from the oxalate-soaked structure is shown as in A. In this conformation, Asp116α is oriented toward the substrate-binding pocket. Structure is shown with the COOM-TPP intermediate in the active site.

Fig. 5.

Solvent access to the OOR active site is gated by the domain III plug loop. (A) Monomer 2 of the oxalate-soaked crystal (shown colored by domain) is superimposed upon monomer 4 from the same crystal (colored white). Domain III in monomer 2, colored yellow, is up to 5 Å closer to the active site than in monomer 4, as indicated by the arrow. The domain III plug loop of monomer 2, colored orange, is ordered and forms a salt bridge through Arg31α to the COOM-TPP intermediate, shown in Inset. (B) The plug loop is ordered in monomer 1 of the oxalate cocrystal. Carboxy-TPP and the proximal [4Fe-4S] cluster are shown buried underneath the protein surface. Domain III is colored yellow. (C) Domain III is rotated away in monomer 2 of the oxalate cocrystal, causing the plug loop to be disordered. Together with Arg31α being rotated away from the active site, a channel to the carboxy-TPP intermediate is visible.

Fig. 6.

Proposed mechanism for oxalate oxidation by OOR. (A) The catalytic cycle showing just TPP and substrate with intermediates numbered. Surrounding panels show proposed interactions of the surrounding protein residues with intermediates (B) 1, (C) 2, (D) 3, and (E) 5. Cartoon boxes indicate the position of the Switch and plug loops, with blue boxes indicating a more positively charged active site (Switch loop in Asp-out conformation), red boxes indicating a more negatively charged active site (Switch loop in Asp-in conformation), and yellow trapezoid indicating plug loop in plug-in conformation. Oxalate binding to the OOR active site and subsequent nucleophilic attack is facilitated by a number of hydrogen bond donating and positively charged residues. After the COOM-TPP intermediate is formed, movement of domain III (plug-in to plug-out) allows Arg31α to swing away from the active site, which, coupled with the Switch loop flipping to the Asp-in conformation, causes the net charge of the active site to become more negative. This change, and especially the interaction between Asp116α and COOM-TPP, may facilitate the first decarboxylation and two subsequent one-electron oxidations. The final deprotonation before the second decarboxylation may be facilitated by Asp116α flipping back out, allowing Gln211α′ to organize a water for hydrogen bonding to the TPP-bound acid.