A designed Copper Histidine-brace enzyme for oxidative depolymerization of polysaccharides as a model of lytic polysaccharide monooxygenase

Author:

Liu Yiwei12ORCID,Harnden Kevin A.1,Van Stappen Casey2ORCID,Dikanov Sergei A.3ORCID,Lu Yi12ORCID

Affiliation:

1. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801

2. Department of Chemistry, University of Texas at Austin, Austin, TX 78712

3. Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61801

Abstract

The “Histidine-brace” (His-brace) copper-binding site, composed of Cu(His) 2 with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV–Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation.

Funder

National Science Foundation

Welch Foundation

HHS | National Institutes of Health

U.S. Department of Energy

Publisher

Proceedings of the National Academy of Sciences

Subject

Multidisciplinary

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