Molecular evolution of hydrogen peroxide degrading enzymes

AbstractFor efficient removal of intra- and/or extracellular hydrogen peroxide by dismutation to harmless dioxygen and water (2H2O2→O2+2H2O), nature designed three metalloenzyme families that differ in oligomeric organization, monomer architecture as well as active site geometry and catalytic residues. Here we report on the updated reconstruction of the molecular phylogeny of these three gene families. Ubiquitous typical (monofunctional) heme catalases are found in all domains of life showing a high structural conservation. Their evolution was directed from large subunit towards small subunit proteins and further to fused proteins where the catalase fold was retained but lost its original functionality. Bifunctional catalase–peroxidases were at the origin of one of the two main heme peroxidase superfamilies (i.e. peroxidase–catalase superfamily) and constitute a protein family predominantly present among eubacteria and archaea, but two evolutionary branches are also found in the eukaryotic world. Non-heme manganese catalases are arelatively small protein family with very old roots only present among bacteria and archaea. Phylogenetic analyses of the three protein families reveal features typical (i) for the evolution of whole genomes as well as (ii) for specific evolutionary events including horizontal gene transfer, paralog formation and gene fusion. As catalases have reached a striking diversity among prokaryotic and eukaryotic pathogens, understanding their phylogenetic and molecular relationship and function will contribute to drug design for prevention of diseases of humans, animals and plants

Structural and mechanistic studies on eukaryotic catalase-peroxidase

Phylogenetische und biochemische Studien zeigten, dass bifunktionelle Katalase-Peroxidasen (KatGs) Teil der Familie I der Peroxidase-Katalase Superfamilie sind und sowohl eine Peroxidase Aktivität als auch eine sehr effiziente Katalase Aktivität aufweisen. Erstmalig konnte eine hochaufgelöste Kristallstruktur einer eukaryotischen KatG im Ruhezustand und in der Oxoeisen(IV) Form präsentiert werden. Im Vergleich zu prokaryotischen KatGs zeigt das extrazelluläre Metalloprotein des Pilzes Magnaporthe oryzae (grisea) einige Besonderheiten, wie zwei konservierte Disulfidbrücken, die die Untereinheiten des homodimeren Proteins verknüpfen. Diese zusätzlichen Quervernetzungen erhöhen sowohl die thermische als auch die konformationelle Stabilität dieser Oxidoreduktase. Neben einem Häm b, tragen KatGs eine einzigartige posttranslationelle Modifikation, die sehr nah am aktiven Zentrum zu finden ist. Drei Aminosäuren, ein Methionin, Tyrosin und ein Tryptophan, sind kovalent miteinander verbunden. Dieses sogenannte kovalente Addukt fungiert als zusätzlicher redox-aktiver Kofaktor und ist essentiell für die Katalase-Aktivität. Diese Arbeit zeigt die wichtige Rolle des Addukts bei der Oxidation von Wasserstoffperoxid und der Freisetzung von Sauerstoff. Zerstörung des intakten Addukts durch Mutagenese führt zur kompletten Beseitigung der Katalase-Aktivität und zur Umwandlung der bifunktionellen KatG zu einem monofunktionellen Enzym. Zusätzlich wurde gezeigt, dass ein konserviertes Arginin, relativ weit weg vom aktiven Zentrum, die Katalase-Aktivität von KatGs zusätzlich moduliert. Röntgenkristallographische Strukturen bei verschiedenenen pH Werten und molekulardynamische Simulationen zeigten eine pH- abhängige Mobilität der Seitenkette. Es konnte nachgewiesen werden, dass dieses Arginin die Reaktivität des Addukts moduliert, indem es die Interaktion des zwischenzeitlich gebildeten Superoxids mit dem Adduktradikal begünstigt. Die Ergebnisse dieser Arbeit konnten in Form eines postulierten Reaktionsmechanismus für die Katalase-Aktivität von KatGs zusammengefasst werden, der alle verfügbaren experimentellen Ergebnisse sowohl von prokaryotischen als auch von eukaryotischen Katalase-Peroxidasen berücksichtigt.Bifunctional catalase-peroxidases (KatGs) have been shown to be part of Family I of the peroxidase-catalase superfamily and exhibit a peroxidatic activity as well as a highly efficient catalatic activity. By utilizing the robust extracellular KatG from the fungus Magnaporthe oryzae (grisea), we present the first high resolution crystal structure of a eukaryotic KatG in its ferric resting state as well as in the oxoiron(IV) state. The structures reveal several novelties compared to prokaryotic KatGs. Importantly, the two subunits of this homodimeric protein are crosswise intertwined by two highly conserved disulphide bridges, enhancing both the thermal and conformational stability of the eukaryotic representative. Beside heme b, KatGs carry a unique posttranslational modification very close to the active site, i.e. the so called covalent adduct autocatalytically formed between the three amino acids methionine, tyrosine and tryptophan (MYW adduct). It acts as an additional redox-active cofactor that is crucial for the catalatic reaction of KatGs. We confirm the essential role of the adduct as a radical site during hydrogen peroxide turnover and demonstrate that disruption of the adduct abolishes the catalase activity and converts the bifunctional enzyme to a monofunctional peroxidase. A conserved arginine residue far from the heme b but close to the covalent adduct additionally modulates the reactivity of the MYW adduct and in consequence the catalatic reaction of KatGs. Structural investigations confirm the pH dependent movement of the arginine side chain pointing either towards the adduct or away. This pH dependent interaction of the arginine with the adduct tyrosine is further supported by molecular dynamics simulations. Altogether, the structural and mechanistic findings allow to propose a mechanism of hydrogen peroxide dismutation that is in line with experimental data from both prokaryotic and eukaryotic catalase-peroxidases.submitted by Bernhard GasselhuberZusammenfassung in deutscher SpracheUniversität für Bodenkultur Wien, Dissertation, 2016OeBB(VLID)193034

Eukaryotic Catalase-Peroxidase: The Role of the Trp-Tyr-Met Adduct in Protein Stability, Substrate Accessibility, and Catalysis of Hydrogen Peroxide Dismutation

Bernhard Gasselhuber et al.© 2015 American Chemical Society. Recently, it was demonstrated that bifunctional catalase-peroxidases (KatGs) are found not only in archaea and bacteria but also in lower eukaryotes. Structural studies and preliminary biochemical data of the secreted KatG from the rice pathogen Magnaporthe grisea (MagKatG2) suggested both similar and novel features when compared to those of the prokaryotic counterparts studied so far. In this work, we demonstrate the role of the autocatalytically formed redox-active Trp140-Tyr273-Met299 adduct of MagKatG2 in (i) the maintenance of the active site architecture, (ii) the catalysis of hydrogen peroxide dismutation, and (iii) the protein stability by comparing wild-type MagKatG2 with the single mutants Trp140Phe, Tyr273Phe, and Met299Ala. The impact of disruption of the covalent bonds between the adduct residues on the spectral signatures and heme cavity architecture was small. By contrast, loss of its integrity converts bifunctional MagKatG2 to a monofunctional peroxidase of significantly reduced thermal stability. It increases the accessibility of ligands due to the increased flexibility of the KatG-typical large loop 1 (LL1), which contributes to the substrate access channel and anchors at the adduct Tyr. We discuss these data with respect to those known from prokaryotic KatGs and in addition present a high-resolution structure of an oxoiron compound of MagKatG2.This project was supported by the Austrian Science Foundation, FWF [Doctoral program BioToP-Biomolecular Technology of Proteins (W1224) and Projects P23855 and P25270]Peer Reviewe

Interaction with the redox cofactor MYW and functional role of a mobile arginine in eukaryotic catalase-peroxidase

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases with an additional posttranslationally formed redox-active Met-Tyr-Trp cofactor that is essential for catalase activity. On the basis of studies of bacterial KatGs, controversial mechanisms of hydrogen peroxide oxidation were proposed. The recent discovery of eukaryotic KatGs with differing pH optima of catalase activity now allows us to scrutinize those postulated reaction mechanisms. In our study, secreted KatG from the fungus Magnaporthe grisea (MagKatG2) was used to analyze the role of a remote KatG-typical mobile arginine that was shown to interact with the Met-Tyr-Trp adduct in a pH-dependent manner in bacterial KatGs. Here we present crystal structures of MagKatG2 at pH 3.0, 5.5, and 7.0 and investigate the mobility of Arg461 by molecular dynamics simulation. Data suggest that at pH ≥4.5 Arg461 mostly interacts with the deprotonated adduct Tyr. Elimination of Arg461 by mutation to Ala slightly increases the thermal stability but does not alter the active site architecture or the kinetics of cyanide binding. However, the variant Arg461Ala lost the wild-type-typical optimum of catalase activity at pH 5.25 (k = 6450 s) but exhibits a broad plateau between pH 4.5 and 7.5 (k = 270 s at pH 5.5). Moreover, significant differences in the kinetics of interconversion of redox intermediates of wild-type and mutant protein mixed with either peroxyacetic acid or hydrogen peroxide are observed. These findings together with published data from bacterial KatGs allow us to propose a role of Arg461 in the HO oxidation reaction of KatG.Peer Reviewe

UDP-sulfoquinovose formation by Sulfolobus acidocaldarius

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD+-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-d-glucose is converted via UDP-4-keto-d-glucose to UDP-d-glucose-5,6-ene, the structure of which was determined by 1H and 13C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-d-glucose and NAD+ could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-d-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested

Eukaryotic Catalase-Peroxidase: The Role of the Trp-Tyr-Met Adduct in Protein Stability, Substrate Accessibility, and Catalysis of Hydrogen Peroxide Dismutation

Recently, it was demonstrated that bifunctional catalase-peroxidases (KatGs) are found not only in archaea and bacteria but also in lower eukaryotes. Structural studies and preliminary biochemical data of the secreted KatG from the rice pathogen <i>Magnaporthe grisea</i> (<i>Mag</i>KatG2) suggested both similar and novel features when compared to those of the prokaryotic counterparts studied so far. In this work, we demonstrate the role of the autocatalytically formed redox-active Trp140-Tyr273-Met299 adduct of <i>Mag</i>KatG2 in (i) the maintenance of the active site architecture, (ii) the catalysis of hydrogen peroxide dismutation, and (iii) the protein stability by comparing wild-type <i>Mag</i>KatG2 with the single mutants Trp140Phe, Tyr273Phe, and Met299Ala. The impact of disruption of the covalent bonds between the adduct residues on the spectral signatures and heme cavity architecture was small. By contrast, loss of its integrity converts bifunctional <i>Mag</i>KatG2 to a monofunctional peroxidase of significantly reduced thermal stability. It increases the accessibility of ligands due to the increased flexibility of the KatG-typical large loop 1 (LL1), which contributes to the substrate access channel and anchors at the adduct Tyr. We discuss these data with respect to those known from prokaryotic KatGs and in addition present a high-resolution structure of an oxoiron compound of <i>Mag</i>KatG2

Eukaryotic extracellular catalase–peroxidase from Magnaporthe grisea – Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV–Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a Kd value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure–function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host–pathogen interaction