ABSTRACT
Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum, EngBF, has a strict substrate preference towards the Gal(1-3)GalNAc glycan O-linked to serine or threonine residues. This glycan is found on proteins such as kappa casein, some mucins, and fetuin.
This thesis addresses various aspects of EngBF catalysis, and is based on three manuscripts focusing on EngBF, which (I) describes an assay for EngBF activity on a glycopeptide substrate, (II) addresses the role of an ordered and conserved water molecule, linking important catalytic residues and (III) describes a mutant variant of EngBF with an expanded substrate spectrum.
We describe steady state kinetics of EngBF catalyzed release of Gal(1-3)GalNAc from a glycopeptide substrate, using an assay developed during this project. This assay is based on the Morgan-Elson colorimetric method, and is specific towards saccharides containing a N-acetylhexosamine, such as GalNAc at the reducing end. We show that this assay is suitable for analyzing EngBF activity towards glycopeptides, and the results obtained indicate the need to consider the leaving group, peptide or small molecule, like para-nitrophenol, when describing a glycoprotein active enzyme, such as EngBF.
Additionally, we investigated the influence on catalysis of several conserved residues, surrounding an ordered water molecule in EngBF. This water molecule links two residues crucial for catalysis, namely Asp-682 and catalytic nucleophile Asp-789. The role in catalysis of Asp-682 remains unclear. The water molecule was surrounded by three other residues, two histidines and an asparagine, all within distance of forming hydrogen bonds with the ordered water molecule. Sequence based alignment showed these residues to be conserved among the characterized Glycoside Hydrolase(GH) family 101 enzymes, and the crystal structures of a
homologous enzyme from Streptococcus pneumoniae, EngSP, contains the water molecule as well as the interacting residues at identical positions. We additionally found the interacting residues to be mostly conserved in a GH129 enzyme, exo-α-N-acetylgalactosaminidase from Bifidobacterium bifidum, NagBb. To address the role of the ordered water molecule in EngBF, we performed amino acid replacements at the residues Asp-682, His-685 and His-718. Kinetic analysis of these mutant variants shows that besides Asp-682 and catalytic nucleophile Asp-789, the network surrounding these residues is also important.
Finally, we report the engineering of an EngBF mutant, with an expanded substrate spectrum, encompassing the sialylated core 1 glycan, Neu5Ac(2-3)Gal(1-3)GalNAc O-linked to serine or threonine residues. Wild type EngBF has s strict substrate preference towards the mucin core 1 glycan, Galβ1-3GalNAc-Ser/Thr. We obtained the expanded substrate spectrum by a single amino acid replacement, E1294K. This mutant variant of EngBF was selected from a molecular docking experiment performed on in-silico generated mutant structures derived from a published structure of EngSP. The E1294K mutant variant of EngBF was active towards the Neu5Ac(2-3)Gal(1-3)GalNAc O-linked glycan on present on fetuin. The activity measured correlated to enzyme concentration and we verified that the product did indeed carry a (2-3) bound sialic