Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.     

Isothermal titration calorimetry uncovers substrate promiscuity of bicupin oxalate oxidase from Ceriporiopsis subvermispora

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔH_app). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H₂O₂ production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.     

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

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.     

Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris

Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2*-) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.     

Immobilization of Bacillus subtilis oxalate decarboxylase on a Zn-IMAC resin

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.     

Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases

Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the nonoxidative reaction but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO₂ and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO₂ from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO₂ using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O₂ using MIMS also estimates the oxidase activity. The use of isotope-labeled substrate (¹³C₂-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product ¹³CO₂ (m/z 45), avoiding inaccuracies due to endogenous ¹²CO₂.     

Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess ≤0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.     

Cloning and sequencing of two Ceriporiopsis subvermispora bicupin oxalate oxidase allelic isoforms: implications for the reaction specificity of oxalate oxidases and decarboxylases

Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess < or =0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.     

Burst kinetics and redox transformations of the active site manganese ion in oxalate oxidase: implications for the catalytic mechanism

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O(2)-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.     

Magnetic resonance and kinetic studies of pyruvate, phosphate dikinase. Interaction of oxalate with the phosphorylated form of the enzyme.

Pyruvate, orthophosphate dikinase (EC 2.7.9.1) carries out its catalytic function in three successive partial reactions, the final step being the reaction of pyruvate with a stable phosphoenzyme intermediate to give phosphoenolpyruvate and free enzyme (Evans, H.J., and Wood, H. G. (1968), Proc. Natl. Acad. Sci. U.S.A. 61, 1448). Interactions of oxalate, a structural analog of enolpyruvate, with the phosphorylated form of the enzyme have been investigated by kinetic inhibition measurements and by magnetic resonance studies of manganous ion complexes with the enzyme. Oxalate inhibits the reaction catalyzed by pyruvate, phosphate dikinase, and the inhibition is linearly competitive with respect to pyruvate. The inhibitor constant for oxalate of 25 mu-M is fourfold lower than the Michaelis constant for pyruvate. The enhancement in the longitudinal relaxation rate of water protons (PRR) which occurs upon binding of Mn(II) to the enzyme has been used to monitor binding of oxalate to Mn(II)-enzyme complexes. PRR titrations indicate that the dissociation constant of oxalate from the Mn(II) complex of the free form of the enzyme is an order of magnitude weaker than the kinetically determined Ki. On the other hand, titrations of solutions which contain the phosphorylated form of the enzyme reveal a much stronger binding of oxalate. Moreover, the strength of oxalate binding to the phosphorylated enzyme is a function both of the species and of the concentration of monovalent cations in the solution. In the presence of Tl+, which has the most favorable activator constant for the final partial reaction, the dissociation constant for oxalate from its complex with the phosphorylated enzyme is less than 1 mu-M. Electron paramagnetic resonance (EPR) spectra for the enzyme-bound Mn(II) are sensitive to structural perturbations which occur upon binding of substrates or of oxalate to the enzyme. The EPR spectrum for the Mn(II)-phosphoenzyme-oxalate species is distinguished from spectra for other complexes of the enzyme by unusually narrow line widths and consequent resolution of fine structure from electronic quadrupole splitting. The narrow lines in the EPR spectrum are indicative of a rigid, pseudocrystalline environment for the bound Mn(II). The magnitude and frequency dependence of the PRR for the Mn(II)-phosphoenzyme-oxalate complex indicate that if any water molecules are bound to the Mn(II), their exchange with the bulk water is severely retarded. The kinetic and magnetic resonance studies support the hypothesis that oxalate mimics the reactive intermediate, enolpyruvate, in a complex with the phosphorylated enzyme which may resemble the structure of the transition state of the final partial reaction.     

Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution

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.     

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

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.     

Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein.

Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The approximately 300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K(m) value for d-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis.     

Structural and spectroscopic studies shed light on the mechanism of oxalate oxidase

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.     

Active site geometry of oxalate decarboxylase from Flammulina velutipes: Role of histidine-coordinated manganese in substrate recognition

Oxalate decarboxylase (OXDC) from the wood-rotting fungus Flammulina velutipes, which catalyzes the conversion of oxalate to formic acid and CO(2) in a single-step reaction, is a duplicated double-domain germin family enzyme. It has agricultural as well as therapeutic importance. We reported earlier the purification and molecular cloning of OXDC. Knowledge-based modeling of the enzyme reveals a beta-barrel core in each of the two domains organized in the hexameric state. A cluster of three histidines suitably juxtaposed to coordinate a divalent metal ion exists in both the domains. Involvement of the two histidine clusters in the catalytic mechanism of the enzyme, possibly through coordination of a metal cofactor, has been hypothesized because all histidine knockout mutants showed total loss of decarboxylase activity. The atomic absorption spectroscopy analysis showed that OXDC contains Mn(2+) at up to 2.5 atoms per subunit. Docking of the oxalate in the active site indicates a similar electrostatic environment around the substrate-binding site in the two domains. We suggest that the histidine coordinated manganese is critical for substrate recognition and is directly involved in the catalysis of the enzyme.     

Crystal structure of 3-hydroxyanthranilic acid 3,4-dioxygenase from Saccharomyces cerevisiae: a special subgroup of the type III extradiol dioxygenases

3-Hydroxyanthranilic acid 3,4-dioxygenase (3HAO) is a non-heme ferrous extradiol dioxygenase in the kynurenine pathway from tryptophan. It catalyzes the conversion of 3-hydroxyanthranilate (HAA) to quinolinic acid (QUIN), an endogenous neurotoxin, via the activation of N-methyl-D-aspartate (NMDA) receptors and the precursor of NAD(+) biosynthesis. The crystal structure of 3HAO from S. cerevisiae at 2.4 A resolution shows it to be a member of the functionally diverse cupin superfamily. The structure represents the first eukaryotic 3HAO to be resolved. The enzyme forms homodimers, with two nickel binding sites per molecule. One of the bound nickel atoms occupies the proposed ferrous-coordinated active site, which is located in a conserved double-strand beta-helix domain. Examination of the structure reveals the participation of a series of residues in catalysis different from other extradiol dioxygenases. Together with two iron-binding residues (His49 and Glu55), Asp120, Asn51, Glu111, and Arg114 form a hydrogen-bonding network; this hydrogen-bond network is key to the catalysis of 3HAO. Residues Arg101, Gln59, and the substrate-binding hydrophobic pocket are crucial for substrate specificity. Structure comparison with 3HAO from Ralstonia metallidurans reveals similarities at the active site and suggests the same catalytic mechanism in prokaryotic and eukaryotic 3HAO. Based on sequence comparison, we suggest that bicupin of human 3HAO is the first example of evolution from a monocupin dimer to bicupin monomer in the diverse cupin superfamilies. Based on the model of the substrate HAA at the active site of Y3HAO, we propose a mechanism of catalysis for 3HAO.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Direct Identification of a Bacterial Manganese(II) Oxidase, the Multicopper Oxidase MnxG, from Spores of Several Different Marine Bacillus Species

Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine Bacillus spores, identified as an Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two Bacillus species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse Bacillus species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase.     

Two-step oxidation of glycerol to glyceric acid catalyzed by the Phanerochaete chrysosporium glyoxal oxidase

Glyoxal oxidase of P. chrysosporium is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H(2)O(2). In addition to known substrates, glycerol is also found to be a substrate for glyoxal oxidase. During enzyme turnover, glyoxal oxidase undergoes a reversible inactivation, probably caused by loss of the active site free radical, resulting in short-lasting enzyme activities and undetectable substrate conversions. Enzyme activity could be extended by including two additional enzymes, horseradish peroxidase and catalase, in addition to a redox chemical activator, such as Mn(III) (or Mn(II)+H(2)O(2)) or hexachloroiridate. Using this three-enzyme system glycerol was converted in glyceric acid in a two-step reaction, with glyceraldehyde as intermediate. A possible operation mechanism is proposed in which the three enzymes would work coordinately allowing to maintain a sustained glyoxal oxidase activity. In the course of its catalytic cycle, glyoxal oxidase alternates between two functional and interconvertible reduced and oxidized forms resulting from a two-electron transfer process. However, glyoxal oxidase can also undergo an one-electron reduction to a catalytically inactive form lacking the active site free radical. Horseradish peroxidase could use glyoxal oxidase-generated H(2)O(2) to oxidize Mn(II) to Mn(III) which, in turn, would reoxidize and reactivate the inactive form of glyoxal oxidase. Catalase would remove the excess of H(2)O(2) generated during the reaction. In spite of the improvement achieved using the three-enzyme system, glyoxal oxidase inactivation still occurred, which resulted in low substrate conversions. Possible causes of inactivation, including end-product inhibition, are discussed.     

Bacillus subtilis YxaG is a novel Fe-containing quercetin 2,3-dioxygenase

The Bacillus subtilis genome contains genes for three hypothetical proteins belonging to the bicupin family, two of which we have previously shown to be Mn(II)-dependent oxalate decarboxylases. We have now shown that the third, YxaG, exhibits quercetin 2,3-dioxygenase activity and that it contains Fe ions. This contrasts with the eukaryotic enzyme which contains a Cu ion. YxaG is the first prokaryotic carbon monoxide-forming enzyme that utilises a flavonol to be characterised and is only the second example of a prokaryotic dioxygenolytic carbon monoxide-forming enzyme known to contain a cofactor. It is proposed to rename the B. subtilis gene qdoI.