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Nigel G J Richards - One of the best experts on this subject based on the ideXlab platform.
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second shell hydrogen bond impacts transition state structure in bacillus subtilis Oxalate Decarboxylase
Biochemistry, 2018Co-Authors: Laurie A Reinhardt, Nigel G J RichardsAbstract:There is considerable interest in how “second-shell” interactions between protein side chains and metal ligands might modulate Mn(II) ion redox properties and reactivity in metalloenzymes. One such Mn-dependent enzyme is Oxalate Decarboxylase (OxDC), which catalyzes the disproportionation of Oxalate monoanion into formate and CO2. Electron paramagnetic resonance (EPR) studies have shown that a mononuclear Mn(III) ion is formed in OxDC during catalytic turnover and that the removal of a hydrogen bond between one of the metal ligands (Glu101) and a conserved, second-shell tryptophan residue (Trp132) gives rise to altered zero-field splitting parameters for the catalytically important Mn(II) ion. We now report heavy-atom kinetic isotope effect measurements on the W132F OxDC variant, which test the hypothesis that the Glu101/Trp132 hydrogen bond modulates the stability of the Mn(III) ion during catalytic turnover. Our results suggest that removing the Glu101/Trp132 hydrogen bond increases the energy of the ox...
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second shell hydrogen bond impacts transition state structure in bacillus subtilis Oxalate Decarboxylase
Biochemistry, 2018Co-Authors: Laurie A Reinhardt, Nigel G J RichardsAbstract:There is considerable interest in how “second-shell” interactions between protein side chains and metal ligands might modulate Mn(II) ion redox properties and reactivity in metalloenzymes. One such Mn-dependent enzyme is Oxalate Decarboxylase (OxDC), which catalyzes the disproportionation of Oxalate monoanion into formate and CO2. Electron paramagnetic resonance (EPR) studies have shown that a mononuclear Mn(III) ion is formed in OxDC during catalytic turnover and that the removal of a hydrogen bond between one of the metal ligands (Glu101) and a conserved, second-shell tryptophan residue (Trp132) gives rise to altered zero-field splitting parameters for the catalytically important Mn(II) ion. We now report heavy-atom kinetic isotope effect measurements on the W132F OxDC variant, which test the hypothesis that the Glu101/Trp132 hydrogen bond modulates the stability of the Mn(III) ion during catalytic turnover. Our results suggest that removing the Glu101/Trp132 hydrogen bond increases the energy of the ox...
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substrate binding mode and molecular basis of a specificity switch in Oxalate Decarboxylase
PMC, 2016Co-Authors: Wen Zhu, Laurie A Reinhardt, David N Silverman, Karen N Allen, Lindsey M Easthon, Steven E Cohen, Nigel G J RichardsAbstract:Oxalate Decarboxylase (OxDC) catalyzes the conversion of Oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC by the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent Oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity, however, has now been investigated using Membrane Inlet Mass Spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of Oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C-C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn (II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from increased solvent access to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 A resolution X-ray crystal structure of a complex between Oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which substrate coordinates the catalytically active Mn ion prior to C-C bond cleavage. The “end-on” conformation of Oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding.
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formation of hexacoordinate mn iii in bacillus subtilis Oxalate Decarboxylase requires catalytic turnover
Biochemistry, 2016Co-Authors: Wen Zhu, Jarett Wilcoxen, David R Britt, Nigel G J RichardsAbstract:Oxalate Decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode electron paramagnetic resonance spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of Oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C–C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species...
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Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase
2016Co-Authors: Wen Zhu, Laurie A Reinhardt, David N Silverman, Karen N Allen, Lindsey M Easthon, Steven E Cohen, Nigel G J RichardsAbstract:Oxalate Decarboxylase (OxDC) catalyzes the conversion of Oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC with the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent Oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of Oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C–C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between Oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C–C bond cleavage. The “end-on” conformation of Oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding
Stephen Bornemann - One of the best experts on this subject based on the ideXlab platform.
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ph dependent structures of the manganese binding sites in Oxalate Decarboxylase as revealed by high field electron paramagnetic resonance
Journal of Physical Chemistry B, 2009Co-Authors: Leandro C Tabares, Laura Bowater, Matthew R Burrell, Jessica Gatjens, Christelle Hureau, Vincent L Pecoraro, Stephen BornemannAbstract:A high-field electron paramagnetic resonance (HFEPR) study of Oxalate Decarboxylase (OxdC) is reported. OxdC breaks down Oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site−1 and −2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site −2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described ...
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the identity of the active site of Oxalate Decarboxylase and the importance of the stability of active site lid conformations
Biochemical Journal, 2007Co-Authors: Victoria J Just, Laura Bowater, Matthew R Burrell, Clare E M Stevenson, David M Lawson, Iain Mcrobbie, Stephen BornemannAbstract:Oxalate Decarboxylase (EC 4.1.1.2) catalyses the conversion of Oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant Oxalate oxidase led to a loss of Decarboxylase activity, but only a slight elevation in the Oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural.
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real time monitoring of the Oxalate Decarboxylase reaction and probing hydron exchange in the product formate using fourier transform infrared spectroscopy
Biochemistry, 2006Co-Authors: Mylrajan Muthusamy, Matthew R Burrell, Roger N F Thorneley, Stephen BornemannAbstract:Oxalate Decarboxylase converts Oxalate to formate and carbon dioxide and uses dioxygen as a cofactor despite the reaction involving no net redox change. We have successfully used Fourier transform infrared spectroscopy to monitor in real time both substrate consumption and product formation for the first time. The assignment of the peaks was confirmed using [(13)C]Oxalate as the substrate. The K(m) for Oxalate determined using this assay was 3.8-fold lower than that estimated from a stopped assay. The infrared assay was also capable of distinguishing between Oxalate Decarboxylase and Oxalate oxidase activity by the lack of formate being produced by the latter. In D(2)O, the product with Oxalate Decarboxylase was C-deuterio formate rather than formate, showing that the source of the hydron was solvent as expected. Large solvent deuterium kinetic isotope effects were observed on V(max) (7.1 +/- 0.3), K(m) for Oxalate (3.9 +/- 0.9), and k(cat)/K(m) (1.8 +/- 0.4) indicative of a proton transfer event during a rate-limiting step. Semiempirical quantum mechanical calculations on the stability of formate-derived species gave an indication of the stability and nature of a likely enzyme-bound formyl radical catalytic intermediate. The capability of the enzyme to bind formate under conditions in which the enzyme is known to be active was determined by electron paramagnetic resonance. However, no enzyme-catalyzed exchange of the C-hydron of formate was observed using the infrared assay, suggesting that a formyl radical intermediate is not accessible in the reverse reaction. This restricts the formation of potentially harmful radical intermediates to the forward reaction.
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sad at home solving the structure of Oxalate Decarboxylase with the anomalous signal from manganese using x ray data collected on a home source
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Clare E M Stevenson, Adam Tanner, Stephen Bornemann, Laura Bowater, David M LawsonAbstract:Oxalate Decarboxylase (OxdC) from Bacillus subtilis is a hexamer containing two manganese ions per 43.6 kDa subunit. A single highly redundant data set collected at a medium resolution of 2 A on an in-house X-ray source was sufficient to solve the structure by the single-wavelength anomalous diffraction (SAD) method using the anomalous signal from the manganese ions. The experimentally phased electron-density map was of high quality, enabling 96% of the amino-acid sequence to be automatically traced using ARP/wARP. Further analysis showed that only half of the original raw data were required for successful structure solution. Manganese currently occurs in approximately 2% of PDB entries. A brief survey suggests that several of these structures could also have been determined using manganese SAD. Moreover, the ability of manganese to substitute for other more commonly occurring divalent metal ions may indicate that the use of Mn SAD could have much wider application.
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a closed conformation of bacillus subtilis Oxalate Decarboxylase oxdc provides evidence for the true identity of the active site
Journal of Biological Chemistry, 2004Co-Authors: Victoria J Just, Adam Tanner, Laura Bowater, Clare E M Stevenson, David M Lawson, Stephen BornemannAbstract: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.
Alexander Angerhofer - One of the best experts on this subject based on the ideXlab platform.
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Oxalate Decarboxylase uses electron hole hopping for catalysis
Journal of Biological Chemistry, 2021Co-Authors: Anthony J Pastore, Umar T. Twahir, Ruijie D Teo, Alvaro Montoya, Matthew J Burg, Steven D Bruner, David N Beratan, Alexander AngerhoferAbstract:The hexameric low-pH stress response enzyme Oxalate Decarboxylase catalyzes the decarboxylation of the Oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 A (intrasubunit) to 21.5 A (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 A. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations.
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the structure of Oxalate Decarboxylase at its active ph
bioRxiv, 2018Co-Authors: Matthew J Burg, Umar T. Twahir, Steven D Bruner, Justin L Goodsell, Alexander AngerhoferAbstract:Abstract Oxalate Decarboxylase catalyzes the redox-neutral unimolecular disproportionation reaction of oxalic acid. The pH maximum for catalysis is ~4.0 and activity is negligible above pH7. Here we report on the first crystal structure of the enzyme in its active pH range at pH4.6, and at a resolution of 1.45 A, the highest to date. The fundamental tertiary and quaternary structure of the enzyme does not change with pH. However, the low pH crystals are heterogeneous containing both a closed and open conformation of a flexible loop region which gates access to the N-terminal active site cavity. Residue E162 in the closed conformation points away from the active-site Mn ion owing to the coordination of a buffer molecule, acetate. Since the quaternary structure of the enzyme appears unaffected by pH many conclusions drawn from the structures taken at high pH remain valid. Density functional theory calculations of the possible binding modes of Oxalate to the N-terminal Mn ion demonstrate that both mono- and bi-dentate coordination modes are possible in the closed conformation with an energetic preference for the bidentate binding mode. The simulations suggest that R92 plays an important role as a guide for positioning the substrate in its catalytically competent orientation. A strong hydrogen bond is seen between the bi-dentate bound substrate and E101, one of the coordinating ligands for the N-terminal Mn ion. This suggests a more direct role of E101 as a transient base during the first step of catalysis.
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redox cycling ph dependence and ligand effects of mn iii in Oxalate Decarboxylase from bacillus subtilis
Biochemistry, 2016Co-Authors: Umar T. Twahir, Andrew Ozarowski, Alexander AngerhoferAbstract:This contribution describes electron paramagnetic resonance (EPR) experiments on Mn(III) in Oxalate Decarboxylase of Bacillus subtilis, an interesting enzyme that catalyzes the redox-neutral dissociation of Oxalate into formate and carbon dioxide. Chemical redox cycling provides strong evidence that both Mn centers can be oxidized, although the N-terminal Mn(II) appears to have the lower reduction potential and is most likely the carrier of the +3 oxidation state under moderate oxidative conditions, in agreement with the general view that it represents the active site. Significantly, Mn(III) was observed in untreated OxDC in succinate and acetate buffers, while it could not be directly observed in citrate buffer. Quantitative analysis showed that up to 16% of the EPR-visible Mn is in the +3 oxidation state at low pH in the presence of succinate buffer. The fine structure and hyperfine structure parameters of Mn(III) are affected by small carboxylate ligands that can enter the active site and have been rec...
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Redox Cycling, pH Dependence, and Ligand Effects of Mn(III) in Oxalate Decarboxylase from Bacillus subtilis
2016Co-Authors: Umar T. Twahir, Andrew Ozarowski, Alexander AngerhoferAbstract:This contribution describes electron paramagnetic resonance (EPR) experiments on Mn(III) in Oxalate Decarboxylase of Bacillus subtilis, an interesting enzyme that catalyzes the redox-neutral dissociation of Oxalate into formate and carbon dioxide. Chemical redox cycling provides strong evidence that both Mn centers can be oxidized, although the N-terminal Mn(II) appears to have the lower reduction potential and is most likely the carrier of the +3 oxidation state under moderate oxidative conditions, in agreement with the general view that it represents the active site. Significantly, Mn(III) was observed in untreated OxDC in succinate and acetate buffers, while it could not be directly observed in citrate buffer. Quantitative analysis showed that up to 16% of the EPR-visible Mn is in the +3 oxidation state at low pH in the presence of succinate buffer. The fine structure and hyperfine structure parameters of Mn(III) are affected by small carboxylate ligands that can enter the active site and have been recorded for formate, acetate, and succinate. The results from a previous report [Zhu, W., et al. (2016) Biochemistry 55, 429–434] could therefore be reinterpreted as evidence of formate-bound Mn(III) after the enzyme is allowed to turn over Oxalate. The pH dependence of the Mn(III) EPR signal compares very well with that of enzymatic activity, providing strong evidence that the catalytic reaction of Oxalate Decarboxylase is driven by Mn(III), which is generated in the presence of dioxygen
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immobilization of bacillus subtilis Oxalate Decarboxylase on a zn imac resin
Biochemistry and biophysics reports, 2015Co-Authors: Umar T. Twahir, Laura Molina, Andrew Ozarowski, Alexander AngerhoferAbstract:Oxalate Decarboxylase, a bicupin enzyme coordinating two essential manganese ions per subunit, catalyzes the decomposition of Oxalate into carbon dioxide and formate in the presence of oxygen. Current efforts to elucidate its catalytic mechanism are focused on EPR studies of the Mn. We report on a new immobilization strategy linking the enzyme's N-terminal His6-tag to a Zn-loaded immobilized metal affinity resin. Activity is lowered somewhat due to the expected crowding effect. High-field EPR spectra of free and immobilized enzyme show that the resin affects the coordination environment of the active site Mn ions only minimally. The immobilized preparation was used to study the effect of varying pH on the same sample. Repeated freeze-thaw cycles lead to break down of the resin beads and some enzyme loss from the sample. However, the EPR signal increases due to higher packing efficiency on the sample column.
Laurie A Reinhardt - One of the best experts on this subject based on the ideXlab platform.
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second shell hydrogen bond impacts transition state structure in bacillus subtilis Oxalate Decarboxylase
Biochemistry, 2018Co-Authors: Laurie A Reinhardt, Nigel G J RichardsAbstract:There is considerable interest in how “second-shell” interactions between protein side chains and metal ligands might modulate Mn(II) ion redox properties and reactivity in metalloenzymes. One such Mn-dependent enzyme is Oxalate Decarboxylase (OxDC), which catalyzes the disproportionation of Oxalate monoanion into formate and CO2. Electron paramagnetic resonance (EPR) studies have shown that a mononuclear Mn(III) ion is formed in OxDC during catalytic turnover and that the removal of a hydrogen bond between one of the metal ligands (Glu101) and a conserved, second-shell tryptophan residue (Trp132) gives rise to altered zero-field splitting parameters for the catalytically important Mn(II) ion. We now report heavy-atom kinetic isotope effect measurements on the W132F OxDC variant, which test the hypothesis that the Glu101/Trp132 hydrogen bond modulates the stability of the Mn(III) ion during catalytic turnover. Our results suggest that removing the Glu101/Trp132 hydrogen bond increases the energy of the ox...
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second shell hydrogen bond impacts transition state structure in bacillus subtilis Oxalate Decarboxylase
Biochemistry, 2018Co-Authors: Laurie A Reinhardt, Nigel G J RichardsAbstract:There is considerable interest in how “second-shell” interactions between protein side chains and metal ligands might modulate Mn(II) ion redox properties and reactivity in metalloenzymes. One such Mn-dependent enzyme is Oxalate Decarboxylase (OxDC), which catalyzes the disproportionation of Oxalate monoanion into formate and CO2. Electron paramagnetic resonance (EPR) studies have shown that a mononuclear Mn(III) ion is formed in OxDC during catalytic turnover and that the removal of a hydrogen bond between one of the metal ligands (Glu101) and a conserved, second-shell tryptophan residue (Trp132) gives rise to altered zero-field splitting parameters for the catalytically important Mn(II) ion. We now report heavy-atom kinetic isotope effect measurements on the W132F OxDC variant, which test the hypothesis that the Glu101/Trp132 hydrogen bond modulates the stability of the Mn(III) ion during catalytic turnover. Our results suggest that removing the Glu101/Trp132 hydrogen bond increases the energy of the ox...
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substrate binding mode and molecular basis of a specificity switch in Oxalate Decarboxylase
PMC, 2016Co-Authors: Wen Zhu, Laurie A Reinhardt, David N Silverman, Karen N Allen, Lindsey M Easthon, Steven E Cohen, Nigel G J RichardsAbstract:Oxalate Decarboxylase (OxDC) catalyzes the conversion of Oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC by the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent Oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity, however, has now been investigated using Membrane Inlet Mass Spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of Oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C-C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn (II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from increased solvent access to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 A resolution X-ray crystal structure of a complex between Oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which substrate coordinates the catalytically active Mn ion prior to C-C bond cleavage. The “end-on” conformation of Oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding.
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Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase
2016Co-Authors: Wen Zhu, Laurie A Reinhardt, David N Silverman, Karen N Allen, Lindsey M Easthon, Steven E Cohen, Nigel G J RichardsAbstract:Oxalate Decarboxylase (OxDC) catalyzes the conversion of Oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC with the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent Oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of Oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C–C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between Oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C–C bond cleavage. The “end-on” conformation of Oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding
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a structural element that facilitates proton coupled electron transfer in Oxalate Decarboxylase
Biochemistry, 2012Co-Authors: Benjamin T Saylor, Alexander Angerhofer, Laurie A Reinhardt, Mithila S Shukla, Linda Nguyen, Wallace W Cleland, Karen N Allen, Nigel G J RichardsAbstract:The conformational properties of an active-site loop segment, defined by residues Ser161-Glu162-Asn163-Ser164, 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 ...
Govindan Sadasivam Selvam - One of the best experts on this subject based on the ideXlab platform.
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in experimental rats
2016Co-Authors: Recombinant Lactobacillus Plantarum Expressing, Ponnusamy Sasikumar, Sivasamy Gomathi, Eldho Paul, Albert Abhishek, Varadaraj Vasudevan, Sundaresan Sasikumar, Oxalate Decarboxylase Urolithiasis, Aswamy Anbazhagan, Govindan Sadasivam SelvamAbstract:and secreting heterologous Oxalate Decarboxylase reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal i
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recombinant lactobacillus plantarum expressing and secreting heterologous Oxalate Decarboxylase prevents renal calcium Oxalate stone deposition in experimental rats
Journal of Biomedical Science, 2014Co-Authors: Ponnusamy Sasikumar, Kolandaswamy Anbazhagan, Sivasamy Gomathi, Eldho Paul, Albert Abhishek, Varadaraj Vasudevan, Sundaresan Sasikumar, Govindan Sadasivam SelvamAbstract:Calcium Oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary Oxalate excretion resulting from decreased intestinal Oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo Oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous Oxalate Decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo Oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium Oxalate diet and 14th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan’s multiple-range test. Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the Oxalate up to 70–77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary Oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V. The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary Oxalate excretion and CaOx crystal deposition by increased intestinal Oxalate degradation.
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genetically engineered lactobacillus plantarum wcfs1 constitutively secreting heterologous Oxalate Decarboxylase and degrading Oxalate under in vitro
Current Microbiology, 2014Co-Authors: Ponnusamy Sasikumar, Kolandaswamy Anbazhagan, Sivasamy Gomathi, Ebenezer A Baby, J Sangeetha, Govindan Sadasivam SelvamAbstract:Hyperoxaluria is a major risk factor for urinary stone disease, where calcium Oxalate (CaOx) is the most prevalent type of kidney stones. Systemic treatments of CaOx kidney stone patients are limited and comprise drawbacks including recurrence of stone formation and kidney damages. In the present work Lactobacillus plantarum (L. plantarum) was engineered to constitutively secrete Oxalate Decarboxylase (OxdC) for the degradation of intestinal Oxalate. The homologous promoter PldhL and signal peptide Lp_0373 of L. plantarum were used for constructing recombinant vector pLdhl0373OxdC. Results showed that homologous promoter PldhL and signal peptide Lp_0373 facilitated the production, secretion, and functional expression of OxdC protein in L. plantarum. SDS-PAGE analysis revealed that 44 kDa protein OxdC was seen exceptionally in the culture supernatant of recombinant L. plantarum (WCFS1OxdC) harboring the plasmid pLdhl0373OxdC.The culture supernatant of L. plantarum WCFS1OxdC showed OxdC activity of 0.06 U/mg of protein, whereas no enzyme activity was observed in the supernatant of the wild type WCFS1 and the recombinant NC8OxdC strains. The purified recombinant OxdC from the WCFS1OxdC strain showed an activity of 19.1 U/mg protein. The recombinant L. plantarum strain secreted 25 % of OxdC protein in the supernatant. The recombinant strain degraded more than 70 % of soluble Oxalate in the culture supernatant. Plasmid segregation analysis revealed that the recombinant strain lost almost 70–89 % of plasmid in 42nd and 84th generation, respectively. In conclusion, recombinant L. plantarum strain containing plasmid pLdhl0373OxdC showed constitutive secretion of bioactive OxdC and also capable of degrading externally available Oxalate under in vitro conditions.
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in vitro degradation of Oxalate by recombinant lactobacillus plantarum expressing heterologous Oxalate Decarboxylase
Journal of Applied Microbiology, 2013Co-Authors: Kolandaswamy Anbazhagan, Ponnusamy Sasikumar, Sivasamy Gomathi, H P Priya, Govindan Sadasivam SelvamAbstract:Aim The aim of the present study is to constitutively express heterologous Oxalate Decarboxylase (OxdC) in Lactobacillus plantarum and to examine its ability to degrade Oxalate in vitro for their future therapy against enteric hyperoxaluria. Method and Results In this study, we generated a recombinant strain of Lb. plantarum to constitutively overexpress B. subtilis Oxalate Decarboxylase (oxdC) using a host lactate dehydrogenase promoter (PldhL). The recombinant Lb. plantarum was able to degrade more than 90% Oxalate compared to 15% by the wild type. In addition, the recombinant strain also had higher tolerance up to 500 mmol l−1 Oxalate. Conclusion We developed a recombinant Lb. plantarum NC8 that constitutively expressed heterologous Oxalate Decarboxylase and degraded Oxalate efficiently under in vitro conditions. Significance and impact of study The long-term aim is to develop an efficient strain for future therapy against oxalosis.
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secretion of biologically active heterologous Oxalate Decarboxylase oxdc in lactobacillus plantarum wcfs1 using homologous signal peptides
BioMed Research International, 2013Co-Authors: Ponnusamy Sasikumar, Kolandaswamy Anbazhagan, Sivasamy Gomathi, Govindan Sadasivam SelvamAbstract:Current treatment options for patients with hyperoxaluria and calcium Oxalate stone diseases are limited and do not always lead to sufficient reduction in urinary Oxalate excretion. Oxalate degrading bacteria have been suggested for degrading intestinal Oxalate for the prevention of calcium Oxalate stone. Here, we reported a recombinant Lactobacillus plantarum WCFS1 (L. plantarum) secreting heterologous Oxalate Decarboxylase (OxdC) that may provide possible therapeutic approach by degrading intestinal Oxalate. The results showed secretion and functional expression of OxdC protein in L. plantarum driven by signal peptides Lp_0373 and Lp_3050. Supernatant of the recombinant strain containing pLp_0373sOxdC and pLp_3050sOxdC showed OxdC activity of 0.05 U/mg and 0.02 U/mg protein, while the purified OxdC from the supernatant showed specific activity of 18.3 U/mg and 17.5 U/mg protein, respectively. The concentration of OxdC protein in the supernatant was 8–12 μg/mL. The recombinant strain showed up to 50% Oxalate reduction in medium containing 10 mM Oxalate. In conclusion, the recombinant L. plantarum harboring pLp_0373sOxdC and pLp_3050sOxdC can express and secrete functional OxdC and degrade Oxalate up to 50% and 30%, respectively.
