Structure-function relationships in catalase-peroxidases
Abstract
In developing ideas of how protein structure modifies heme reactivity, class I peroxidases are an exciting field of research. Despite striking sequence homologies, class I members (yeast cytochrome c peroxidase, ascorbate peroxidase and catalase-peroxidases) exhibit dramatic differences in catalytic activity and substrate specificity. Cytochrome c peroxidase (CCP) is unusual in that it uses another protein (cytochrome c) as a redox partner, whereas ascorbate peroxidases (APXs) prefer the anion ascorbate as electron donor. Both CCP and APX do not exhibit substantial catalase activity. By contrast, multifunctional catalase-peroxidases (KatGs) exhibit a catalase activity comparable to monofunctional catalases in addition to function as peroxidase of broad-specificity. But the structural basis for this extraordinary reactivity is unknown. In order to elucidate structure-function relationships of catalase-peroxidases, in this project we plan to perform comprehensive mutational analysis in combination with kinetic and spectroscopic (UV-Vis, resonance Raman and electronic paramagnetic) investigations. Three recombinant catalase-peroxidases will be investigated in this project. Since the three-dimensional structure of KatG is unknown crystallization studies of recombinant holoproteins and truncated proteins which lack parts at the N- and C-terminal region as well as parts of the KatG-typical flexible loops are planned. Based on multiple sequence alignment and secondary structure prediction, mutational analysis will be performed. Besides manipulations at the highly conserved active site, the project is focused on KatG specific insertions, three of them are typical for KatGs showing highly conserved sequence patterns. A hypothesis for the roles of these KatG specific insertions is presented and (based on data from molecular modeling in combination with site-direted fluorescence labeling) site-directed mutagenesis experiments are planned and recombinant variants are produced. The spectral and kinetic features of the variants will be investigated by steady-state and transient-state kinetic measurements. Information about iron coordination and spin states, hydrogen bonding networks in ferric and ferrous proteins, the nature (e.g. existence, location and role of protein radicals) and reactivity of the redox intermediates as well as the influence of the protein matrix on heme substituent orientation and heme side residue geometries and distances will be investigated by resonance Raman and EPR spectroscopy. In the proposal a hypothesis for the possible electronic structure of KatG intermediates is presented and should be tested within this project.
Publications
Engineering the proximal heme cavity of catalase-peroxidase.
Autoren: Jakopitsch, C; Regelsberger, G; Furtmüller, PG; Rüker, F; Peschek, GA; Obinger, C Jahr: 2002
Journal articles
Understanding the Heme Cavity of Catalase-Peroxidase via Site-Directed Mutagenesis and Resonance Raman Spectroscopy
Autoren: Smulevich, G., Santoni, E., Heering, H. A., Indiani, C., Jakopitsch, C., Regelsberger, G., and Obinger, C. Jahr: 2002
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Distal site aspartate is essential in the catalase activity of catalase-peroxidases.
Autoren: Jakopitsch, C; Auer, M; Regelsberger, G; Jantschko, W; Furtmüller, PG; Rüker, F; Obinger, C Jahr: 2003
Journal articles
Identification of the radical intermediates formed by the catalase-peroxidase of Synechocystis PCC6803: A multifrequency EPR spectroscopy study combined with isotope labelling and site directed mutagenese
Autoren: Ivancich, A., Jakopitsch, C., and Obinger, C. Jahr: 2003
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Towards understanding the catalatic activity of peroxidases and catalases?
Autoren: Jakopitsch, C., Schmuckenschlager, F., Zehner, F., Wanasinghe, A., Furtmüller, P. G., Obinger, C. Jahr: 2004
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:The role in the stability of the heme architecture of selected catalase-peroxidase mutants. An overview of a spectroscopic study.
Autoren: Droghetti, E., Jakopitsch, C., Obinger, C., Smulevich, G Jahr: 2005
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:The role of the unusual crosslinked distal site Trp-Tyr-Met adduct in catalysis of bifunctional catalase-peroxidase
Autoren: Jakopitsch, C., Vlasits, J., Ivancich, A., Obinger, C., Jahr: 2005
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Trapping the spectral features of the redox intermediates in the catalatic cycle of catalase-peroxidase
Autoren: Jakopitsch, C., Vlasits, J., Obinger, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Identification of Trp106 as the tryptophanyl radical intermediate in Synechocystis PCC6803 catalase-peroxidase by multifrequency Electron Paramagnetic Resonance spectroscopy.
Autoren: Jakopitsch, C; Obinger, C; Un, S; Ivancich, A Jahr: 2006
Journal articles
Redox thermodynamics of the Fe(III)/Fe(II) couple in wild-type heme peroxidases and in their CN-adducts
Autoren: Bellei, M., Jakopitsch, C., Battistuzzi, G., Sola, M., Obinger, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Protein-based radical intermediates in bifunctional heme peroxidases
Autoren: Ivancich, A., Un, S., Obinger, C., Loewen, P. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide.
Autoren: Jakopitsch, C; Spalteholz, H; Furtmüller, PG; Arnhold, J; Obinger, C; Jahr: 2008
Journal articles
Modulation of catalytic functions in heme enzymes by posttranslational modifications of redox sites
Autoren: Obinger, C. Jahr: 2011
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Chlorite as oxidant and reductant of heme peroxidases: a mechanistic study
Autoren: Furtmüller, P.G., Jakopitsch, C., Spalteholz, H., Arnhold, J., Obinger, C. Jahr: 2007
Journal articles
external links and characteristics of the publication:Do we really understand the catalatic reactivity?
Autoren: Jakopitsch, C; Petutschnig, G; Zehner, F; Ruker, F; Furtmuller, PG; Obinger, C Jahr: 2003
Journal articles
Interaction with the Redox Cofactor MYW and Functional Role of a Mobile Arginine in Eukaryotic Catalase-Peroxidase
Autoren: Gasselhuber, B; Graf, MMH; Jakopitsch, C; Zamocky, M; Nicolussi, A; Furtmuller, PG; Oostenbrink, C; Carpena, X; Obinger, C Jahr: 2016
Journal articles
New insights into the heme cavity structure of catalase-peroxidase: a spectroscopic approach to the recombinant synechocystis enzyme and selected distal cavity mutants.
Autoren: Heering, HA; Indiani, C; Regelsberger, G; Jakopitsch, C; Obinger, C; Smulevich, G Jahr: 2002
Journal articles
The 2.0A resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase.
Autoren: Atzenhofer, W; Regelsberger, G; Jacob, U; Peschek, G; Furtmüller, P; Huber, R; Obinger, C Jahr: 2002
Journal articles
The catalytic role of the distal site asparagine-histidine couple in catalase-peroxidases.
Autoren: Jakopitsch, C; Auer, M; Regelsberger, G; Jantschko, W; Furtmüller, PG; Rüker, F; Obinger, C Jahr: 2003
Journal articles
Spectroscopic and kinetic investigations of selected mutants of recombinant catalase-peroxidase from synecocystis
Autoren: Santoni, E., Jakopitsch, C., Heering, H.A., Indiani, C., Regelsberger, G., Obinger, C., Smulevich, G., Jahr: 2003
Chapter in collected volumes
external links and characteristics of the publication:Total conversion of bifunctional catalase-peroxidase (KatG) to monofunctional peroxidase by exchange of a conserved distal side tyrosine.
Autoren: Jakopitsch, C; Auer, M; Ivancich, A; Rüker, F; Furtmüller, PG; Obinger, C Jahr: 2003
Journal articles
Distal side tryptophan, tyrosine and methionine in catalase-peroxidases are covalently linked in solution.
Autoren: Jakopitsch, C; Kolarich, D; Petutschnig, G; Furtmüller, PG; Obinger, C Jahr: 2003
Journal articles
Comparison between catalase-peroxidase and cytochrom c peroxidase: A resonance raman study
Autoren: Santoni, E., Jakopitsch, C., Obinger, C., and Smulevich, G. Jahr: 2003
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Engineering catalase-peroxidases: The structural basis of the activity of catalase-peroxidases.
Autoren: Petutschnig, G., Jakopitsch, C., Auer, M., Rüker, F., Furtmüller, P. G., and Obinger, C. Jahr: 2003
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Heme peroxidase biochemistry--facts and perspectives.
Autoren: Obinger, C Jahr: 2010
Journal articles
Mechanism of hydrogen peroxide oxidation in catalase-peroxidases
Autoren: Jakopitsch, C., Vlasits, J., Obinger, C. Jahr: 2010
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Protein-based radicals in the catalase-peroxidase of synechocystis PCC6803: a multifrequency EPR investigation of wild-type and variants on the environment of the heme active site.
Autoren: Ivancich, A; Jakopitsch, C; Auer, M; Un, S; Obinger, C Jahr: 2003
Journal articles
Insights into catalase-peroxidase by electronic absorption, resonance Raman spectroscopy, and site-directed mutagenesis
Autoren: Smulevich, G., Santoni, E., Jakopitsch, C., Obinger, C. Jahr: 2004
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Comparison between catalase-peroxidase and cytochrome c peroxidase. The role of the hydrogen-bond networks for protein stability and catalysis.
Autoren: Santoni, E; Jakopitsch, C; Obinger, C; Smulevich, G Jahr: 2004
Journal articles
Spectral and kinetic studies on redox intermediates in heme peroxidases from both superfamilies
Autoren: Obinger, C., Furtmüller, P.G., Zederbauer, M., Jakopitsch, C. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:The role of selected residues in the heme architecture of catalase-peroxidases
Autoren: Droghetti, E., Jakopitsch, C., Obinger, C., Smulevich, G. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
Probing the role of the peculiar Trp-Tyr-Met adduct in the heme pocket of bifunctional catalase-peroxidases
Autoren: Jakopitsch, C., Vlasits, J., Ivancich, A., Obinger, C. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Probing the structure and bifunctionality of catalase-peroxidase (KatG).
Autoren: Smulevich, G; Jakopitsch, C; Droghetti, E; Obinger, C Jahr: 2006
Journal articles
Redox thermodynamics of the ferric-ferrous couple of wild-type synechocystis KatG and KatG(Y249F).
Autoren: Bellei, M; Jakopitsch, C; Battistuzzi, G; Sola, M; Obinger, C Jahr: 2006
Journal articles
The catalatic activity of monofunctional catalases and bifunctional catalase-peroxidases follows different mechanisms
Autoren: Obinger, C., Vlasits, J., Jakopitsch, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Investigation of the catalytic mechanism of Synechocystis catalase-peroxidase with doubly O18-labeled hydrogen peroxide
Autoren: Vlasits, J., Jakopitsch, C., Holubar, P., Obinger, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Identification of the unique site (Trp106) for the formation of the [Fe(IV)=O Trp.] intermediate in Synechocystis PCC6803 KatG and the (distal heme side) extended H-bonding network with a crucial role in catalase activity vy using multifrequency (9-285 GHz) EPR spectroscopy
Autoren: Jakopitsch, C., Obinger, C., Un, S., Ivancich, A. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Towards understanding the role of protein-based radical intermediates as alternative oxidation sites in mono- and bifunctional peroxidases (KatGs)
Autoren: Loewen, P. C., Obinger, C., Un, S., Ivancich, A. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Detoxification of hydrogen peroxide in cyanobacteria
Autoren: Bernroitner, M., Jakopitsch, C., Obinger, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Probing the reaction of three distinct catalase-peroxidases with peroxides
Autoren: Jakopitsch, C., Wiseman, B., Vlasits, J., Obinger, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Hydrogen peroxide oxidation by catalase-peroxidase follows a non-scrambling mechanism.
Autoren: Vlasits, J; Jakopitsch, C; Schwanninger, M; Holubar, P; Obinger, C; Jahr: 2007
Journal articles
Catalases
Autoren: Obinger, C., Furtmüller, P. G., Zamocky, M. Jahr: 2012
Chapter in collected volumes
Investigation of the catalytic mechanism of Synechocystis catalase-peroxidase with doubly O18-labeled hydrogen peroxide
Autoren: Vlasits, J., Jakopitsch, C., Holubar, P., Obinger, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:A GC-MS study on the catalatic mechanism of catalase-peroxidase
Autoren: Vlasits, J., Jakopitsch, C., Holubar, P., Obinger, C. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Mechanisms of catalase activity of heme peroxidases.
Autoren: Vlasits, J; Jakopitsch, C; Bernroitner, M; Zamocky, M; Furtmüller, PG; Obinger, C; Jahr: 2010
Journal articles
Redox thermodynamic of the Fe3+/Fe2+ couple in wild-type and mutated heme peroxidases
Autoren: Battistuzzi, G., Bellei, M., Jakopitsch, C., Vlasits, J., Furtmüller, P.G., Sola, M., Obinger, C. Jahr: 2007
Journal articles
external links and characteristics of the publication:Fungal catalase-peroxidases - a novel group of bifunctional oxidoreductases
Autoren: Zamocky, M., Jakopitsch, C., Vlasits, J., Obinger, C. Jahr: 2007
Journal articles
external links and characteristics of the publication:
Project staff
Christian Obinger
Univ.Prof. Mag.rer.nat. Dr.rer.nat. Christian Obinger
christian.obinger@boku.ac.at
Tel: +43 1 47654-10011, 77273
Project Leader
01.01.2002 - 31.12.2004
Christa Jakopitsch
Mag.Dr. Christa Jakopitsch
Tel: +43 1 47654-33061
Project Staff
01.01.2002 - 31.12.2004
Martina Paumann-Page
Dipl.-Ing. Dr.rer.nat. Martina Paumann-Page
Project Staff
01.01.2002 - 31.12.2004
BOKU partners
External partners
Institute of Chemistry
Giulietta Smulevich
partner
Section Bioenergetics, Biophysics of Oxidative Stress_x000D_ CEA-Saclay, France
Anabella Ivancich
partner