One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography Marcus I. Gibsona,1, Percival Yang-Ting Chena, Aileen C. Johnsona,2, Elizabeth Pierceb,3, Mehmet Canb, Stephen W. Ragsdaleb, and Catherine L. Drennana,c,d,4 aDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109; cHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139; and dDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved November 30, 2015 (received for review September 21, 2015) Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (Fig. 1A) (10). This OOR, discovered in Moorella thermoacetica (OOR) metabolizes oxalate, generating two molecules of CO2 and (11, 12), feeds both carbon dioxide and low-potential electrons two low-potential electrons, thus providing both the carbon and into the Wood−Ljungdahl pathway for acetogenesis, allowing reducing equivalents for operation of the Wood−Ljungdahl path- M. thermoacetica to grow exclusively on oxalate—a boon to this way of acetogenesis. Here we present structures of OOR in which anaerobic bacterium commonly found in soil, which is often rich two different reaction intermediate bound states have been trap- in oxalate (13). Like oxalyl-CoA decarboxylase, OOR uses TPP ped: the covalent adducts between TPP and oxalate and between as its catalytic cofactor, but differs in that it does not require TPP and CO2. These structures, along with the previously deter- CoA to activate oxalate, and it uses a chain of [4Fe-4S] clusters mined structure of substrate-free OOR, allow us to visualize how to store and transfer the electrons captured from the C−C bond. active site rearrangements can drive catalysis. Our results suggest OOR belongs to a larger superfamily of microbial enzymes, that OOR operates via a bait-and-switch mechanism, attracting known as 2-oxoacid:ferredoxin oxidoreductases (OFORs), which substrate into the active site through the presence of positively use TPP and [4Fe-4S] clusters to oxidize pyruvate and other charged and polar residues, and then altering the electrostatic cellular building blocks (14, 15). The best characterized family environment through loop and side chain movements to drive member is pyruvate:ferredoxin oxidoreductase (PFOR), for which catalysis. This simple but elegant mechanism explains how oxa- X-ray structures of substrate-free and substrate-bound forms (SI late, a molecule that humans and most animals cannot break Appendix,Fig.S1B) as well as some biochemical data are available down, can be used for growth by acetogenic bacteria. for the Desulfovibrio africanus enzyme (Da PFOR) (16–19), and more biochemical and spectroscopic data are available for the oxalate | oxidoreductase | carbon dioxide | thiamine pyrophosphate M. thermoacetica enzyme (20–23). PFOR catalyzes the reversible cleavage of pyruvate to form CO2, acetyl-CoA, and two electrons xalate is a difficult molecule to metabolize, so much so that that are capable of reducing low-potential ferredoxins (Fig. 1B). In Ohumans and most animals lack the capacity to break it this reaction, TPP acts as a nucleophile and forms a covalent down. This inability to metabolize oxalate is associated with adduct with pyruvate to initiate the subsequent decarboxylation. chronic renal disease in humans, some of the manifestations of Although we assume the same will be true for TPP-dependent which are painful kidney stones, crystalline arthritis, and total – OOR, oxalate is a very different substrate than pyruvate and the kidney failure (1 3). Plants, fungi, and some Gram-positive other 2-oxoacid substrates of the OFOR superfamily, all of which bacteria have evolved oxygen-dependent pathways for metabo- are characterized by a ketone immediately adjacent to a carboxylic lizing oxalate with the help of manganese-dependent enzymes in the cupin family of proteins (4, 5). These aerobic enzymes, ox- alate oxidase and oxalate decarboxylase, take advantage of the Significance propensity of oxalate to ligate metal ions to form a Mn oxalate complex, following which dioxygen is used to generate an oxalate- The microbial Wood−Ljungdahl pathway is the biological equiv- based radical species that causes rapid decarboxylation. The re- alent of the Monsanto process, responsible for converting duced products of these enzymes are hydrogen peroxide in the greenhouse gas CO2 into acetate. In addition to CO2, this microbial case of the oxidase and formate in the case of the decarboxylase. pathway requires low-potential electrons. The recently discovered Before 2010, only one enzyme pathway was known to catalyze the oxalate oxidoreductase produces both low-potential electrons degradation of oxalic acid in the absence of dioxygen: oxalyl-CoA and CO2 through the oxidation of oxalate. Here our structural decarboxylase (6). This enzyme is best known for its role in the data allow us to visualize intermediates along the reaction cycle metabolism of Oxalobacter formigenes, a bacterium that colonizes that have not been previously described, providing insight into the human gut and has been the subject of research into the role of the molecular mechanism by which oxalate is metabolized. the human gut microbiome in preventing, and possibly treating, – Author contributions: M.I.G., S.W.R., and C.L.D. designed research; M.I.G., P.Y.-T.C., A.C.J., human renal disease (7 9). As its name implies, however, oxalyl- E.P., and M.C. performed research; M.I.G., P.Y.-T.C., S.W.R., and C.L.D. analyzed data; and CoA decarboxylase first requires the activation of oxalate by CoA, M.I.G. and C.L.D. wrote the paper. a feat that is accomplished by a formyl-CoA transferase. This acti- The authors declare no conflict of interest. vated oxalate, featuring a thioester in place of a carboxylate, is This article is a PNAS Direct Submission. susceptible to nucleophilic attack by thiamin pyrophosphate (TPP; Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, SI Appendix,Fig.S1A), which is the catalytic cofactor in this an- www.pdb.org (PDB ID codes 5EXD and 5EXE). aerobic enzyme. The end product of this reaction is formate, which 1Present address: Department of Chemistry, Princeton University, Princeton, NJ 08540. can either enter cellular metabolism or be pumped out of the cell by 2Present address: Emory University School of Medicine, Atlanta, GA 30307. a formate:oxalate antiporter to generate ATP (8). 3Present address: Physical Science Department, Southern Utah University, Cedar City, Recently, an oxalate oxidoreductase (OOR) was reported that UT 84720. performs a previously unknown metabolic transformation of 4To whom correspondence should be addressed. Email: [email protected]. oxalate by splitting it into two molecules of carbon dioxide and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. recovering two reducing equivalents for use in cellular processes 1073/pnas.1518537113/-/DCSupplemental. 320–325 | PNAS | January 12, 2016 | vol. 113 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1518537113 Downloaded by guest on September 28, 2021 (25). Note that the Greek symbols after the residue number in- dicate the OOR chain ID (see SI Appendix, Fig. S1D). The structure presented here of OOR with oxalate covalently attached to TPP is consistent with this hypothesis. Arg31α occupies an analogous position to Thr31, and substrate interactions with Arg109α (OOR)/Arg114 (PFOR) and the imine group of the TPP iminopyr- imidine are also conserved (Fig. 3 B and E). Additionally, Asn143β (OOR)/Asn996 (PFOR) is in a similar position to hydrogen bond Fig. 1. OOR and PFOR catalyze the oxidation of oxalate and pyruvate, re- with the bound substrate, although this 3.1-Å interaction appears to spectively, reducing two ferredoxin equivalents in the process. (A) The re- action scheme for OOR. (B) The reaction scheme for PFOR. be with O2 of the bound oxalate, whereas, in PFOR, Asn996 inter- acts with the carboxylate group of the substrate (Fig. 3 B and E). To facilitate hydrogen-bonding interactions, rotation of the ethyl linker acid. Oxalate, on the other hand, is a diprotic acid and carries two of TPP (that links the thiazole ring to the pyrophosphate moiety) negative charges at neutral pH (24), and is therefore less ame- changes the angle of the thiazole plane, tilting the bound substrate nable to nucleophilic attack by a TPP cofactor. toward the various hydrogen bond donors. This ethyl linker rotation To probe how OOR was adapted to act on oxalate, we pre- is akin to previously described TPP flexibility in PFOR (19), and it is viously solved the structure of OOR from M. thermoacetica in its not observed in any active sites in which oxalate is not bound. resting state (25). Structural analysis revealed that OOR is a Unexpectedly, a close contact with the COOM intermediate is α – dimer of trimers, (αγβ)2, with each αγβ forming a catalytic unit also made by Asp116 , which is part of a loop from residues 107 that is composed of five domains (SI Appendix, Fig. S1 C and D). 121 of chain α that helps form the substrate-binding pocket. In the The active site, which houses the TPP and one of the three [4Fe- previously reported structure (Fig. 4A), Asp116α points away from 4S] clusters, appears to be more polar than that found in PFOR, theactivesiteandPhe117α points toward where substrate would consistent with OOR’s unique substrate (25). Here, we further bind (25) (an Asp-out conformation). However, in each of the four interrogate substrate binding to OOR through soaking and active sites of the oxalate-soaked structure, we see this situation cocrystallization experiments with oxalate. In addition to reversed, with Asp116α rotated about 150° to point toward the identifying residues responsible for activating oxalate, these carboxylate group of oxalate, Phe117α rotated about 90°, and the structures allow us to consider the molecular mechanism phenyl group swung away from the active site (Asp-in conforma- responsible for conversion of oxalate to CO2 and release of tion) (Fig.