First Crystal Structure of a Fungal High-redox Potential Dye-decolorizing Peroxidase: SUBSTRATE INTERACTION SITES AND LONG-RANGE ELECTRON TRANSFER

Eric Strittmatter,Christiane Liers,René Ullrich,Sabrina Wachter,Martin Hofrichter,Dietmar A Plattner,Klaus Piontek

doi:10.1074/jbc.m112.400176

Abstract

Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.1 Å resolution. The mostly helical structure also shows a β-sheet motif typical for DyPs and Cld (chlorite dismutase)-related structures and includes the complete polypeptide chain. At the distal side of the heme molecule, a flexible aspartate residue (Asp-168) plays a key role in catalysis. It guides incoming hydrogen peroxide toward the heme iron and mediates proton rearrangement in the process of Compound I formation. Afterward, its side chain changes its conformation, now pointing toward the protein backbone. We propose an extended functionality of Asp-168, which acts like a gatekeeper by altering the width of the heme cavity access channel. Chemical modifications of potentially redox-active amino acids show that a tyrosine is involved in substrate interaction. Using spin-trapping experiments, a transient radical on the surface-exposed Tyr-337 was identified as the oxidation site for bulky substrates. A possible long-range electron transfer pathway from the surface of the enzyme to the redox cofactor (heme) is discussed.

Highlights

Identification of Substrate Oxidation Sites—To explore the possibility of small substrates being oxidized directly in the heme cavity, we tentatively modeled a Compound I state of AauDyPI
Because no post-translational changes that would directly indicate such redox-active residues could be detected during model building, we could not identify a surface-exposed substrate interaction site from the crystal structure
To address this issue experimentally, in a first step, we measured enzyme activities after chemically modifying tyrosine and tryptophan residues

Summary

Background

Results: Based on the crystal structure of a fungal DyP, the conformational flexibility of Asp-168 is elucidated. Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. Peroxidases (EC 1.11.1) are ubiquitous enzymes that catalyze the oxidative conversion of various compounds utilizing hydrogen peroxide (H2O2) as electron acceptor. A division of this heterogenic group led to the formation of four families, one of which consist of the dye-decolorizing peroxidases or DyPs3 (EC 1.11.1.19) [1]. DyPs turned out to be phylogenetically as well as structurally unrelated to all hitherto described peroxidase families. The enzyme responsible for the decolorization process was characterized as a heme peroxidase of unusual chemical properties. Enzymatic assays on a fungal DyP from the basidiomycete Auricularia auricula-judae (AauDyP, formerly labeled “AjP”) have been reported in a

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION