Structural studies of Lytic Polysaccharide Monooxygenases

Tobias Tandrup

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

Abstract

Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes discovered within the last ten years, which oxidatively degrade recalcitrant biomass, making chains available for further degradation by hydrolytic enzymes. LPMOs show great potential as a key component in formulation of efficient enzyme cocktails for biomass conversion, and as such, for the production of bioethanol.
Very little experimental information on the interactions of LPMOs with their substrates was available until recently. A combined biochemical, crystallographic and spectroscopic study elucidated the interactions between a family AA9 LPMO of the filamentous fungus Lentinus similis (LsAA9_A) and cello-oligosaccharides. Similar interactions were observed through NMR studies for a family AA9 LPMO of Neurospora crassa and glucans.
In this thesis, I present studies focused on comparing substrate interactions and activity of LsAA9_A and a related LPMO from Collariella virescens (CvAA9_A). I studied substrate interactions using X-ray crystallography, and was able to solve complex structures for the CvAA9_A LPMO. I have investigated the use of Differential Scanning Fluorimetry as a guide to make complex structures with LPMOs and their substrates, and investigate enzyme stability. Enzyme stability and plasticity was further explored using Molecular Dynamics simulations on LsAA9_A, CvAA9_A and the family AA13 LPMO from Aspergillus oryzae (AoAA13). A long-term goal is to elucidate the enzymatic mechanism of LPMOs. To that extent, I studied the first step in the LPMO mechanism – transition from Cu(II) to Cu(I). I did this using X-ray photoreduction, which revealed geometric changes around the Cu-site. A co-substrate of LPMOs has been discovered to be either O2 or H2O2. In this work, I have experimented with crystal oxygenation for chemically reduced and cello-oligosaccharide substrate soaked crystals. During my studies I also aimed to determine the first neutron structure of an LPMO in complex with cello-oligosaccharide to further elucidate the structural determinants of LPMO substrate binding. Finally, I participated in characterising a new family of LPMO-like proteins named X325. These proteins are structurally similar to LPMOs, but do not display oxidative activity.
OriginalsprogEngelsk
ForlagDepartment of Chemistry, Faculty of Science, University of Copenhagen
Antal sider244
StatusUdgivet - 2021

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