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Weston, Thomas (2025) FGE-catalysed modification of CAZymes: A new approach for monitoring protein-cellulose interactions. PhD thesis, University of Nottingham.

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Abstract

Fossil fuels dominate the global energy sector and are considered the primary reason for the rise in greenhouse gases. Renewables represent a sustainable alternative yet lack investment in infrastructure. Biofuels are one alternative that may allow us to achieve the goals outlined in the 2015 Paris agreement. 2nd Generation biofuels use waste biomass from primary feedstock production. Lignocellulosic biomass is the most abundant biomass source globally, making it a promising energy source. Organisms across nature are known to produce enzymes capable of degrading polysaccharides, which are exploited in commercial bioethanol synthesis. Despite this little is understood about how these enzymes bind to extended cellulose surfaces. Expanding this knowledge may allow us to exploit these enzymes industrially.

Techniques like Nuclear Magnetic Resonance (NMR) spectroscopy and X-ray crystallography cannot be applied to insoluble polysaccharides which are often the natural substrates of these enzymes. Electron Paramagnetic Resonance (EPR) spectroscopy can use solids and has been used to characterise the binding of lytic polysaccharide monooxygenases (LPMOs) to cellulose surfaces. Unlike LPMOs, glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs), lack the paramagnetic species required for this technique.

Traditional protein labelling methods typically utilise long flexible linkers and are not site specific. Formylglycine generating enzyme (FGE) represents the opportunity to introduce a small, versatile aldehyde functionality in a site-specific manner which can be reacted with a variety of aminooxy and hydrazine functionalised reagents.

Here we present the methods to site selectively modify CBMs and GHs using FGE which can then be reacted with a hydrazone functionalised ligand scaffold, capable of chelating copper. In turn, this will provide the paramagnetic species required to expand the EPR methodology to other classes of CAZymes.

This work outlines the synthesis and characterisation of a ligand scaffold, its reactivity and its copper coordinating abilities. Using commercially available aldehydes we developed a library of Schiff base ligands to test the reactivity of the ligand scaffold. Using NMR and X-ray crystallography confirmed that the ligand reacts with aldehydes at the hydrazone moiety. We then investigated these ligands copper coordinating properties using UV-vis, electron diffraction, EPR and X-ray crystallography. UV-vis data suggested the ligand binds copper in a 2:1 stoichiometry (ligand:copper). However, resolving the structure of the complex by electron diffraction showed a 1:1 stoichiometry.

A form of FGE was cloned, expressed and purified. Its activity was confirmed using a synthetic peptide substrate, where we detected the aldehyde product by mass spectrometry and reverse-phase liquid chromatography. The reactivity of the modified peptide with the ligand scaffold was demonstrated using similar techniques. We also showed that FGE prefers hydrophobic residues within its recognition sequence, using a series of synthetic peptides with varying sidechains. It was proposed that this preference is likely due to the topology of the active site only accommodating short hydrophobic side chains.

The methods for introducing the site-specific recognition motif into a recombinant CBM of Clostridium thermocellum are outlined alongside a coexpression method to obtain a modified CBM with an aldehyde functionality. We were able to show that the mutant CBM folded into its natural β-sandwich structure and binds to crystalline cellulose. We were able to coexpress the mutant CBM wit FGE and detect the formylglycine residue using a fluorescent probe in combination with gel electrophoresis. The aldehyde product was reacted with the ligand scaffold which we were able to detect using mass spectrometry methods. EPR spectroscopy of the modified CBM suggests an adventitious binding site within the proteins structure. The EPR data shows that the coordination environment of this adventitious binding site is likely composed of oxygen and nitrogen atoms. It was hypothesised that the copper is coordinating with a calcium binding site that stabilises the secondary structure however, this needs experimental confirmation.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Ciano, Luisa O'Neill, Ellis Thomas, Neil
Keywords:	Biofuels, Formylglycine generating enzyme (FGE), Carbohydrate active enzymes CAZymes
Subjects:	Q Science > QD Chemistry > QD241 Organic chemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Chemistry
Item ID:	82311
Depositing User:	Weston, Thomas
Date Deposited:	12 Dec 2025 04:40
Last Modified:	12 Dec 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/82311

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