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 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.
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Bernhard
B
Gasselhuber
Institut de Biologia Molecular (IBMB-CSIC) , Parc Cientific de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
Xavi
X
Carpena
Department of Material Sciences and Process Engineering, Institute for Molecular Modeling and Simulation, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Michael M H
MM
Graf
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Katharina F
KF
Pirker
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Andrea
A
Nicolussi
Department of Material Sciences and Process Engineering, Institute for Molecular Modeling and Simulation, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Axel
A
Sündermann
Department for Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna , Campus Biocenter 5, A-1030 Vienna, Austria.
Stefan
S
Hofbauer
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.|Institute of Molecular Biology, Slovak Academy of Sciences , Dubravska cesta 21, SK-84551 Bratislava, Slovakia.
Marcel
M
Zamocky
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Paul G
PG
Furtmüller
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Christa
C
Jakopitsch
Department of Material Sciences and Process Engineering, Institute for Molecular Modeling and Simulation, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Chris
C
Oostenbrink
Institut de Biologia Molecular (IBMB-CSIC) , Parc Cientific de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
Ignacio
I
Fita
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Christian
C
Obinger
D002374:Catalase / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D005057:Eukaryotic Cells / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D020082:Magnaporthe / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014363:Tryptamines / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D014363:Tryptamines; C030820:tryptamine; D014443:Tyrosine; D008715:Methionine; D006861:Hydrogen Peroxide; D002374:Catalase; D009195:Peroxidase