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Structural and biochemical studies enlighten the unspecific peroxygenase from hypoxylon sp. EC38 as an efficient oxidative biocatalyst

DOI: 10.1021/acscatal.1c03065 DOI Help

Authors: Laura Rotilio (University of Pavia) , Alexander Swoboda (University of Graz) , Katharina Ebner (Graz University of Technology) , Claudia Rinnofner (raz University of Technology) , Anton Glieder (Graz University of Technology) , Wolfgang Kroutil (University of Graz) , Andrea Mattevi (University of Pavia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: September 2021
Diamond Proposal Number(s): 20221

Open Access Open Access

Abstract: Unspecific peroxygenases (UPOs) are glycosylated fungal enzymes that can selectively oxidize C–H bonds. UPOs employ hydrogen peroxide as the oxygen donor and reductant. With such an easy-to-handle cosubstrate and without the need for a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from Hypoxylon sp. EC38 (HspUPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris. The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. HspUPO is a versatile catalyst performing various reactions, such as the oxidation of prim- and sec-alcohols, epoxidations, and hydroxylations. Semipreparative biotransformations were demonstrated for the nonenantioselective oxidation of racemic 1-phenylethanol rac-1b (TON = 13 000), giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated yield. HspUPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with substrates of variable polarities. The structural investigation of several protein–ligand complexes revealed that the active site of HspUPO is accessible to molecules of varying bulkiness with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, HspUPO is an exploitable candidate for peroxygenase-based biocatalysis.

Journal Keywords: substrate recognition; protein tunnels; biocatalytic oxidation; monooxygenase; peroxygenase

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography

Other Facilities: Swiss Light Synchrotron; European Synchrotron Radiation Facility

Added On: 06/09/2021 08:58

Documents:
acscatal.1c03065.pdf

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)