Engineering improved specificity and activity into oral bacterial sialidases for glycan biotechnology applications

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To understand and develop orally derived sialidases for the glycobiology industry.Study design and methodology1-Structural understanding of PG0352 and NanH (Yr 1-2)Structural information on PG0352 and NanH sialidases is needed given their novel activity and CBM domains to understand mode of action and direct engineering. -Using protocols established by GS and JR we will purify and crystallise full NanH and PG0352 and their isolated CBM domains (where sialloconjugates will not be cleaved) in apo- and ligand-bound forms (e.g. 3/6- S-lactose, SLeA/X, S-Tn, DANA). We have conditions for crystallization of the NanH CBM (GS, JR) suggesting good potential for other constructs. To increase chances of novel ligand bound information we will produce inactive versions (FRIP mutations) to promote ligand binding.-In parallel we will undertake in-depth bioinformatic and modeling studies of NanH and PG0352 based on pdb-hit information and that gained here to predict residues that might improve specificity.2-Mining of the oral microbiome for novel sialidases (Yr 1-2)Preliminary screening of the Human Oral Microbiome Database resource reveals a plethora of sialidases with novel potential. - We will mine this resource and a range of oral metagenome datasets for novel sialidases that we will produce as codon optimized (novel IP) clones that we will purify and assess for activity using our established glyconjugate (GS) and glycoprotein (DS) desialylation assays using standard biochemical methods (Thiobarbiturate assay (GS)) or derivatised HPLC or MS based methods (DS- to be performed at Ludger). These will also enter crystallisation trials to increase the opportunities for successful structure solution.3- Engineering sialidases for specificity and activity improvements (Yr 2-3)-Based on modeling, structural information (1) and on the conservation of the catalytic Arginine triad and characteristic FRIP domain and Asp-boxes we will target residues surrounding the active site (e.g. Trp212 and Tyr119 of the T. cruzi enzyme) and ligand-binding residues of the CBM contacting ligands. The likelihood that we will be able to achieve this is increased by our novel approach examining the CBM domains in isolation and also in the use of inactive mutants that should still bind ligands.