The mechanism, structural and functional specificity of sulfoglycosidases from glycoside hydrolase 20 family

Dong, Mochen 2024. The mechanism, structural and functional specificity of sulfoglycosidases from glycoside hydrolase 20 family. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Mucin glycan sulfation is an important post-translational modification that supports the barrier function of mucin in maintaining the health of human gastrointestinal, respiratory, and reproductive tracts. Bacteria must overcome this sulfation barrier to utilise the mucin as a nutrient source for themselves and other symbiotic bacteria. Sulfatases have been recognised as a common mechanism for mucin desulfation, thereby enabling glycosidases to hydrolyse the desulfated sugars. However, the discovery of sulfoglycosidases indicates an alternative mechanism which could function with sulfatases or potentially mitigate the requirement of them. This discovery reveals a novel pathway by which the microbiota can access sulfoglycans. Exo-acting sulfoglycosidases from human microbiota are glycosidases involved in removing of Nacetyl-6-O-sulfo-D-glucosamine (6S-GlcNAc) from host glycans, playing a crucial role in health and disease. However, the mechanisms underlying their ability to recognize 6-sulfate groups remain unclear. In the first part of this project, we used kinetic analysis and protein structures revealed by our previous work to identify the catalytically important amino acids involved in the recognition and cleavage of 6S-GlcNAc in BbhII from Bifidobacteriam bifidum, Bt4394 from Bacteroides thetaiotaomicron, and SGL from Prevotella sp. These 6S-GlcNAcases do not cleave 6P-GlcNAc, possibly due to the different charges and protonation states of the phosphate group. Currently, only a limited number of sulfoglycosidases have been identified, and traditional approaches for identifying new sulfoglycosidases are laborious. To address this, in the second part of this thesis, we integrated bioinformatics approaches with structures derived from AI-driven tools like AlphaFold 3 to screen metagenomics for unknown GH enzymes towards 6S-GlcNAc. Using this method, we identified two potential new sulfate recognition sequences from GH20 family enzymes: WGPYYINR from the WG enzyme of Prevotella sp. and YGPYYINR from the YG enzyme of Alloprevotella sp. Functional analysis confirmed that these two wild-type enzymes are highly effective 6S-GlcNAcases, with their sulfate recognition motifs identified through mutagenesis and kinetic studies. Subsequently, we employed the bioinformatics EFI-SSN webtool to identify nine additional 6S-GlcNAcases bearing these newly found binding motifs within the GH20 family. Furthermore, we characterised the previously discovered F3- ORF26 enzyme from Phocaeicola dorei, elucidating serine and asparagine as evolutionarily selected hydrogen bond donors involved in sulfate binding. This comprehensive analysis led to the discovery of new 6S-GlcNAcases through the EFI-SSN search, which were biochemically validated

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Chemistry
Funders:	China Scholarship Council (202006280011)
Date of First Compliant Deposit:	25 February 2025
Last Modified:	26 Feb 2025 09:44
URI:	https://orca.cardiff.ac.uk/id/eprint/176485

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