Structural and Biochemical Studies Enlighten the Unspecific Peroxygenase from Hypoxylon sp. EC38 as an Efficient Oxidative Biocatalyst

Laura Rotilio,Alexander Swoboda,Anton Glieder,Claudia Rinnofner,Andrea Mattevi,Wolfgang Kroutil,Katharina Ebner

doi:10.1021/acscatal.1c03065

Laura Rotilio, Alexander Swoboda + Show 5 more

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https://doi.org/10.1021/acscatal.1c03065

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Abstract

Unspecific peroxygenases (UPO) are glycosylated fungal enzymes that can selectively oxidize C-H bonds. UPOs employ hydrogen peroxide as oxygen donor and reductant. With such an easy-to-handle co-substrate and without the need of 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. Semi-preparative biotransformations were demonstrated for the non-enantioselective oxidation of racemic 1-phenylethanol rac -1b (TON = 13000), 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 small-to-medium size 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 and polarity 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.

Highlights

The C−H bond is relatively inert, and its oxidation is one of the most challenging conversions in chemistry.[1−5] The cytochromeP450s have been historically considered as a attractive group of enzymes to perform oxidative reactions targeting C−H groups.[6]
A number of strategies have been described to improve the challenging expression of these enzymes.[35−39] We identified a highly promising novel unspecific peroxygenase from Hypoxylon sp
We found that HspUPO is optimally active at neutral pH with indole, 5-nitro-1,3-benzodioxole, and benzyl alcohol, whereas more acidic pH values are optimal for the conversions of ABTS, 3,4-dimethoxybenzyl alcohol, and 2,6dimethoxyphenol (Figure 3A)

Summary

Introduction

The C−H bond is relatively inert, and its oxidation is one of the most challenging conversions in chemistry.[1−5] The cytochromeP450s have been historically considered as a attractive group of enzymes to perform oxidative reactions targeting C−H groups.[6]. HspUPO activities were determined by absorbance-based methods using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), indole, 2,6-dimethoxyphenol, 5-nitro-1,3benzodioxole, 3,4-dimethoxybenzyl alcohol, and benzyl alcohol as substrates.

Results

Conclusion