Design and synthesis of chemical tools for the study of family GH99 and GH76 glycoside hydrolases that process n-linked glycans

Abstract

© 2015 Dr. Zalihe HakkiN-linked glycans have significant roles in protein folding, stability, and function. Their aberrant expression is implicated in diseases including cancer and viral infection. Considerable effort has been expended over the years in the search for inhibitors that may interfere with the enzymes involved in their biosynthesis. The focus of many studies has been on inhibiting the enzymes involved in the early processing steps of N-linked glycans, and in particular the ER resident glycosidases. However targeting these enzymes has yielded limited success largely due to the existence of a unique enzyme, a Golgi-resident glycoside hydrolase termed endo-α-1,2-mannosidase. This family GH99 enzyme provides a ‘back-up’ pathway to the biosynthesis of mature N-linked glycans. The first part of this thesis investigates the fundamental features of this poorly understood enzyme using rationally-designed chemical tools and bacterial orthologs of endo-α-1,2-mannosidase from Bacteroides spp. Using the activated substrate α-Glc-1,3-Man-F the enzyme is determined to act with retention of stereochemistry. The first X-ray structure of any GH99 enzyme is solved and complexes with the established endo-α-1,2-mannosidase inhibitor α-Glc-1,3-Man-DMJ and herein synthesized α-Glc-1,3-IFG are used to structurally define the active site of the enzyme. The inhibitor α-Glc-1,3-IFG is instrumental in revealing the absence of a candidate nucleophilic residue and forms the basis for the proposal of an unusual mechanism for family GH99, which is proposed to proceed through a 1,2-anhydro intermediate. Preliminary studies with α-Glc-1,3-IFG also demonstrate that endo-α-1,2-mannosidase is a viable anti-viral target. The subsequent chapters detail the development of a ‘blocked’ derivative of α-Glc-1,3-IFG as a potential selective inhibitor of endo-α-1,2-mannosidase for use in cellular studies. Some work is also detailed towards the development of a dye-labelled α-Glc-1,3-IFG derivative for diagnostic purposes. The thesis proceeds to examine the role of bacterial endo-α-1,2-mannosidase orthologs in bacteria. Mammalian N-linked glycans are structurally-related to yeast α-mannans and it is speculated that bacterial endo-α-1,2-mannosidase may have a role in the degradation of yeast α-mannans. A representative yeast mannan fragment α-Man-1,3-α-Man-1,2-α-Man-1,2-α-Man-OMe is synthesized and shown to be a substrate of bacterial endo-α-1,2-mannosidase. Further the epimerically-related substrates α-Glc-1,3-α-Man-methylumbelliferone and α-Man-1,3-α-Man-methylumbelliferone reveal a ten-fold preference of the bacterial enzyme to process D-manno configured substrates. On the basis of these results, an improved inhibitor, α-Man-1,3-IFG is developed. Comparison of X-ray crystal structures of α-Glc-1,3-IFG and α-Man-1,3-IFG reveal a Trp residue that confers favourable binding of D-manno configured substrates and inhibitors. The final part of this thesis entails efforts to better understand GH76 endo-α-1,6-mannanases, another poorly understood mannosidase family that is present exclusively in bacteria and fungi. In fungi, these enzymes are speculated to be involved in the cross-linking of the cell glycophosphatidylinositol (GPI)-anchored glycoproteins into the fungal cell wall and in bacteria they are proposed to act as hydrolytic enzymes facilitating the degradation of yeast α-mannans. Although previous structures of GH76 have been reported, nothing was known about the catalytic mechanism and conformational itinerary of the enzyme due to a lack of substrates and inhibitors. Our X-ray structures of Bacillus circulans GH76 in complex with substrates and rationally-designed inhibitors proved instrumental in obtaining insights into the active site, mechanism and conformational itinerary of a representative GH76 enzyme for the first time. Through the use of p-nitrophenyl α-1,6-mannobioside, GH76 is shown to be retaining enzyme, supporting its role as a transglycosidase in cross-linking GPI anchors to fungal cell walls. The complex of BcGH76 with the inhibitor α-Man-1,6-IFG reveals two catalytic Asp residues that are likely to facilitate a conventional double displacement retaining mechanism. GH76 is also shown to be the first mannosidase to distort the IFG moiety of α-Man-1,6-IFG to an energetically unfavourable boat conformation, suggesting this conformation has special significance. X-ray structures and QM/MM simulations suggest that GH76 likely operates through a OS2 ↔ B2,5‡ ↔ 1S5 conformational itinerary