Publications

• Enzymatic oxalate decarboxylation in Aspergillus niger. II. Hydrogen peroxide formation and other characteristics of the oxalate decarboxylase.

  Emiliani E., Riera B.

  Biochim Biophys Acta 167:414-421(1968) [PubMed] [EuropePMC]

• Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis.

  Svedruzic D., Liu Y., Reinhardt L.A., Wroclawska E., Cleland W.W., Richards N.G.

  Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured ... >> More

  Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies. << Less

  Arch Biochem Biophys 464:36-47(2007) [PubMed] [EuropePMC]

• Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution.

  Anand R., Dorrestein P.C., Kinsland C., Begley T.P., Ealick S.E.

  Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-po ... >> More

  Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known. << Less

  Biochemistry 41:7659-7669(2002) [PubMed] [EuropePMC]

• Multifrequency EPR studies on the Mn(II) centers of oxalate decarboxylase.

  Angerhofer A., Moomaw E.W., Garcia-Rubio I., Ozarowski A., Krzystek J., Weber R.T., Richards N.G.

  Oxalate decarboxylase from Bacillus subtilis is composed of two cupin domains, each of which contains a Mn(II) ion coordinated by four identical conserved residues. The similarity between the two Mn(II) sites has precluded previous attempts to distinguish them spectroscopically and complicated eff ... >> More

  Oxalate decarboxylase from Bacillus subtilis is composed of two cupin domains, each of which contains a Mn(II) ion coordinated by four identical conserved residues. The similarity between the two Mn(II) sites has precluded previous attempts to distinguish them spectroscopically and complicated efforts to understand the catalytic mechanism. A multifrequency cw-EPR approach has now enabled us to show that the two Mn ions can be distinguished on the basis of their differing fine structure parameters and to observe that acetate and formate bind to Mn(II) in only one of the two sites. The EPR evidence is consistent with the hypothesis that this Mn-binding site is located in the N-terminal domain, in agreement with predictions based on a recent X-ray structure of the enzyme. << Less

  J Phys Chem B 111:5043-5046(2007) [PubMed] [EuropePMC]

• Bacillus subtilis YvrK is an acid-induced oxalate decarboxylase.

  Tanner A., Bornemann S.

  Bacillus subtilis has been shown to express a cytosolic oxalate decarboxylase (EC 4.1.1.2). The enzyme was induced in acidic growth media, particularly at pH 5.0, but not by oxalate. The enzyme was purified, and N-terminal sequencing identified the protein to be encoded by yvrK. The role of the fi ... >> More

  Bacillus subtilis has been shown to express a cytosolic oxalate decarboxylase (EC 4.1.1.2). The enzyme was induced in acidic growth media, particularly at pH 5.0, but not by oxalate. The enzyme was purified, and N-terminal sequencing identified the protein to be encoded by yvrK. The role of the first oxalate decarboxylase to be identified in a prokaryote is discussed. << Less

  J. Bacteriol. 182:5271-5273(2000) [PubMed] [EuropePMC]

• Oxalate decarboxylase requires manganese and dioxygen for activity. Overexpression and characterization of Bacillus subtilis YvrK and YoaN.

  Tanner A., Bowater L., Fairhurst S.A., Bornemann S.

  The Bacillus subtilis oxalate decarboxylase (EC ), YvrK, converts oxalate to formate and CO(2). YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalat ... >> More

  The Bacillus subtilis oxalate decarboxylase (EC ), YvrK, converts oxalate to formate and CO(2). YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manganese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H(2)O(2); and oxalate-dependent, H(2)O(2)-independent dye oxidations. The oxalate decarboxylase activity of purified YvrK was O(2)-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A = 9.5 millitesla) of the main g = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O(2) are cofactors that act together as a two-electron sink during catalysis. << Less

  J. Biol. Chem. 276:43627-43634(2001) [PubMed] [EuropePMC]

• Heavy atom isotope effects on the reaction catalyzed by the oxalate decarboxylase from Bacillus subtilis.

  Reinhardt L.A., Svedruzic D., Chang C.H., Cleland W.W., Richards N.G.

  Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and r ... >> More

  Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity. The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated. Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B. subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669). We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC. At pH 4.2, the OxDC-catalyzed formation of formate and CO(2) have normal (13)C isotope effects of 1.5% +/-0.1% and 0.5% +/-0.1%, respectively, while the (18)O isotope effect on the formation of formate is 1.1% +/-0.2% normal. Similarly at pH 5.7, the production of formate and CO(2) exhibits normal (13)C isotope effects of 1.9% +/- 0.1% and 0.8% +/-0.1%, respectively, and the (18)O isotope effect on the formation of formate is 1.0% +/-0.2% normal. The (18)O isotope effect on the formation of CO(2), however, 0.7% +/-0.2%, is inverse at pH 5.7. These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion. Subsequent reduction and protonation of this intermediate then gives formate. << Less

  J Am Chem Soc 125:1244-1252(2003) [PubMed] [EuropePMC]

• A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site.

  Just V.J., Stevenson C.E., Bowater L., Tanner A., Lawson D.M., Bornemann S.

  Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radic ... >> More

  Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain. << Less

  J Biol Chem 279:19867-19874(2004) [PubMed] [EuropePMC]

• Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid.

  Burrell M.R., Just V.J., Bowater L., Fairhurst S.A., Requena L., Lawson D.M., Bornemann S.

  Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share com ... >> More

  Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase. << Less

  Biochemistry 46:12327-12336(2007) [PubMed] [EuropePMC]

• A structural element that facilitates proton-coupled electron transfer in oxalate decarboxylase.

  Saylor B.T., Reinhardt L.A., Lu Z., Shukla M.S., Nguyen L., Cleland W.W., Angerhofer A., Allen K.N., Richards N.G.

  The conformational properties of an active-site loop segment, defined by residues Ser(161)-Glu(162)-Asn(163)-Ser(164), have been shown to be important for modulating the intrinsic reactivity of Mn(II) in the active site of Bacillus subtilis oxalate decarboxylase. We now detail the functional and s ... >> More

  The conformational properties of an active-site loop segment, defined by residues Ser(161)-Glu(162)-Asn(163)-Ser(164), have been shown to be important for modulating the intrinsic reactivity of Mn(II) in the active site of Bacillus subtilis oxalate decarboxylase. We now detail the functional and structural consequences of removing a conserved Arg/Thr hydrogen-bonding interaction by site-specific mutagenesis. Hence, substitution of Thr-165 by a valine residue gives an OxDC variant (T165V) that exhibits impaired catalytic activity. Heavy-atom isotope effect measurements, in combination with the X-ray crystal structure of the T165V OxDC variant, demonstrate that the conserved Arg/Thr hydrogen bond is important for correctly locating the side chain of Glu-162, which mediates a proton-coupled electron transfer (PCET) step prior to decarboxylation in the catalytically competent form of OxDC. In addition, we show that the T165V OxDC variant exhibits a lower level of oxalate consumption per dioxygen molecule, consistent with the predictions of recent spin-trapping experiments [Imaram et al. (2011) Free Radicals Biol. Med. 50, 1009-1015]. This finding implies that dioxygen might participate as a reversible electron sink in two putative PCET steps and is not merely used to generate a protein-based radical or oxidized metal center. << Less

  Biochemistry 51:2911-2920(2012) [PubMed] [EuropePMC]