Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases.

Tobias Tandrup,Cristina Hernández-Rollán,Katja Salomon Johansen,Morten J Bjerrum,Raushan K Singh,Johan Ørskov Ipsen,Morten H H Nørholm,Kristian Erik Høpfner Frandsen,Leila Lo Leggio,Sebastian J Muderspach,Sanchari Banerjee

doi:10.3390/biom12020194

Abstract

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Highlights

Lytic Polysaccharide Monooxygenases (LPMOs) are a recently discovered group of copper metalloenzymes that depolymerize recalcitrant polysaccharides like lignocellulose and chitin by oxidative cleavage of the glycosidic bonds
Extensive research is being carried out to understand the mechanism of the LPMO catalysis for effective industrial applications [7]
Our present study aims to investigate the protonation state of the His at the 2nd coordination sphere from the AA9 LPMO structures

Summary

Introduction

Lytic Polysaccharide Monooxygenases (LPMOs) are a recently discovered group of copper metalloenzymes that depolymerize recalcitrant polysaccharides like lignocellulose and chitin by oxidative cleavage of the glycosidic bonds. LPMOs have been shown to boost the activities of other carbohydrate active enzymes, breaking down large crystalline/insoluble polysaccharides into glucans [1,2,3]. These sugars can be fermented to bioethanol, a renewable form of energy [4]. The presence of LPMOs or predicted LPMO-encoding genes has been established in many organisms, including microbes, fungi, algae, plants and insects [7]. Their complete role in all the organisms is far from understood, Biomolecules 2022, 12, 194.

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