Abstract
A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN− molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.
Highlights
The social amoeba Dictyostelium discoideum is unusual among eukaryotes in having both unicellular and multicellular stages [1]
In the case of Dictyostelium DyPA, both the aspartate and arginine residue on the distal face are in an optimal position (Figure 6B,C) to take on a catalytic role during compound I formation upon H2 O2 addition
Potential substrate-binding pockets preenergy transfer (LRET) sites of Dictyostelium DyPA. (A) Potential substrate-binding pockets predicted dicted by POCASA. (B,C) Two solvent channels leading to the distal face of Dictyostelium DyPA
Summary
The social amoeba Dictyostelium discoideum is unusual among eukaryotes in having both unicellular and multicellular stages [1]. Dictyostelium discoideum cells are frequently found as an abundant component of the microflora in the upper layer of soil and on decaying organic material [2]. We tested the catalytic activity of Dictyostelium DyPA with a range of different organic substrates and characterized its ferric-heme microenvironment and the formation of catalytic intermediates by UV-Vis, EPR, and time-resolved stopped-flow spectroscopy. X-ray structures of Dictyostelium DyPA in complex with activated oxygen alone and together with veratryl alcohol, as well as the structure of the complex with the competitive inhibitor cyanide, provide detailed insight into the substrate access channel, active site residues, and movement of the DXXDG motif during the formation of compound I, with Asp149 functioning as an acid-base catalyst at low pH [7,17].
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