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Mutational and structural analysis of an ancestral fungal dye‐decolorizing peroxidase

DOI: 10.1111/febs.15687 DOI Help

Authors: Ulises A. Zitare (University of Groningen; Universidad de Buenos Aires and CONICET) , Mohamed H. Habib (University of Groningen; Cairo University) , Henriette Rozeboom (University of Groningen) , Maria L. Mascotti (University of Groningen; Universidad Nacional de San Luis) , Smilja Todorovic (Universidade Nova de Lisboa) , Marco W. Fraaije (University of Groningen)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: The Febs Journal , VOL 41

State: Published (Approved)
Published: January 2021
Diamond Proposal Number(s): 19800

Open Access Open Access

Abstract: Dye‐decolorizing peroxidases (DyPs) constitute a superfamily of heme‐containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A‐C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D‐type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D‐type DyP ancestor, AncDyPD‐b1. Expression of AncDyPD‐b1 in Escherichia coli results in large amounts of a heme‐containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV‐Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady‐state kinetics and the crystal structure, reveal fine pH‐dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long‐range electron transfer process.

Journal Keywords: ancestral sequence reconstruction; crystal structure; D‐type DyP; dye‐decolorizing peroxidase; heme coordination

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Documents:
febs.15687.pdf