Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).
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The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site. (+info)
Striking activation of oxidative enzymes suspended in nonaqueous media.
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The catalytic activity of four lyophilized oxidative enzymes-horseradish peroxidase, soybean peroxidase, Caldariomyces fumago chloroperoxidase, and mushroom polyphenol oxidase-is much lower when directly suspended in organic solvents containing little water than when they are introduced into the same largely nonaqueous media by first dissolving them in water and then diluting with anhydrous solvents. The lower the water content of the medium, the greater this discrepancy becomes. The mechanism of this phenomenon was found to arise from reversible denaturation of the oxidases on lyophilization: because of its conformational rigidity, the denatured enzyme exhibits very limited activity when directly suspended in largely nonaqueous media but renatures and thus yields much higher activity if first redissolved in water. Two independent means were discovered for dramatically minimizing the lyophilization-induced inactivation, both involving the addition of certain types of excipients to the aqueous enzyme solution before lyophilization. The first group of excipients consists of phenolic and aniline substrates as well as other hydrophobic compounds; these presumably bind to the hydrophobic pocket of the enzyme active site, thereby preventing its collapse during dehydration. The second group consists of general lyoprotectants such as polyols and polyethylen glycol that apparently preserve the overall enzyme structure during dehydration. The activation effects of such excipients can reach into the tens and hundreds of fold. Moreover, the activations afforded by the two excipient groups are additive, resulting in up to a complete protection against lyophilization-induced inactivation when representatives of the two are present together. (+info)
Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue.
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Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme. (+info)
Peroxidase and phosphatase activity of active-site mutants of vanadium chloroperoxidase from the fungus Curvularia inaequalis. Implications for the catalytic mechanisms.
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Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics. (+info)
X-ray structures of a novel acid phosphatase from Escherichia blattae and its complex with the transition-state analog molybdate.
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The structure of Escherichia blattae non-specific acid phosphatase (EB-NSAP) has been determined at 1.9 A resolution with a bound sulfate marking the phosphate-binding site. The enzyme is a 150 kDa homohexamer. EB-NSAP shares a conserved sequence motif not only with several lipid phosphatases and the mammalian glucose-6-phosphatases, but also with the vanadium-containing chloroperoxidase (CPO) of Curvularia inaequalis. Comparison of the crystal structures of EB-NSAP and CPO reveals striking similarity in the active site structures. In addition, the topology of the EB-NSAP core shows considerable similarity to the fold of the active site containing part of the monomeric 67 kDa CPO, despite the lack of further sequence identity. These two enzymes are apparently related by divergent evolution. We have also determined the crystal structure of EB-NSAP complexed with the transition-state analog molybdate. Structural comparison of the native enzyme and the enzyme-molybdate complex reveals that the side-chain of His150, a putative catalytic residue, moves toward the molybdate so that it forms a hydrogen bond with the metal oxyanion when the molybdenum forms a covalent bond with NE2 of His189. (+info)
Prochiral selectivity in H(2)O(2)-promoted oxidation of arylalkanols catalysed by chloroperoxidase. The role of the interactions between the OH group and the amino-acid residues in the enzyme active site.
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The H(2)O(2)-promoted oxidations of (R)-[alpha-(2)H(1)]-and (S)-[alpha-(2)H(1)]-arylalkanols catalysed by chloroperoxidase (CPO) from Caldariomyces fumago have been investigated. It has been found that with (R)-[alpha-(2)H(1)]-alcohols, the oxidation involves almost exclusively the cleavage of the C-H bond, whereas in the case of the oxidation of (S)-[alpha-(2)H(1)]-alcohols, the C-D bond is preferentially broken. These results clearly indicate that the reactions of corresponding undeuterated arylalkanols are characterized by a high prochiral selectivity, involving the cleavage of the pro-S C-H bond. This prochiral selectivity is poorly influenced by the electronic effect of ring substituents, whereas it decreases with the length of the carbon lateral chain, in the order: benzyl alcohol > 2-phenylethanol > 3-phenylpropanol. Molecular binding studies showed that the main factor directing the docking of the substrate in such a specific orientation in the enzyme active site is the interaction between the alcoholic OH group and the residue Glu183. This interaction is likely to drive both the stereochemistry and the regiochemistry of these reactions. A bifurcated hydrogen bond involving the OH group, the carboxylate oxygen of Glu183 and the oxoferryl oxygen might also be operating. (+info)
Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme.
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The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases. (+info)
Roles of water in heme peroxidase and catalase mechanisms.
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A water molecule is coproduced with the Compound I intermediate in the reactions of native heme peroxidases and catalases with hydrogen peroxide. As a result of water release/rebinding from/to the coproduct formation site the Compound I intermediate may exist in two forms: a "wet" form, Compound I(H(2)O), in which a water molecule is present at or near the site of coproduct water formation, and Compound I, in which the coproduct water formation site is "dry." It is postulated that the absence or presence of a water molecule at this site provides the structural basis for a redox pathway switching mechanism, such that the transition states for 2-electron equivalent reduction of Compound I intermediates are accessible in the dry form, but that in the wet form only 1-electron equivalent processes are possible, unless release of water can be stimulated. This concept provides the basis of a general mechanism in which the classical functional distinction between catalases and peroxidases, as well as the more complex behavior observed in halide oxidation and halogenation reactions, appear as particular cases in which variations in the degree of retention of water at the coproduct formation site influence Compound I reactivity. (+info)