On the reactivity and selectivity of donor glycosides in glycochemistry and glycobiology

The processes of glycosidic bond formation and destruction are a central theme in glycochemistry and glycobiology, and form the basis of the research described in this Thesis. In the first part, studies towards the stereoselective construction of two complex bacterial oligosaccharide fragments are described. These fragments contain mannuronic acid residues connected through a beta-linkage, which is amongst the most challenging linkages to construct synthetically. In the second part, the use of mannuronic acid building blocks in the automated synthesis of alginates using solid-phase chemistry is presented. Using a second-generation carbohydrate synthesizer, alginate fragments up to 12 residues were assembled with high stereoselectivity. Using the same automated set-up, fragments of hyaluronic acid of up to 15 residues were synthesized. In the third part, 2-deoxy-2-fluoroglucosides are investigated as activity-based probes for glucocerebrosidase, a retaining beta-glucosidase enzyme.NWO, TI PharmaUBL - phd migration 201

A β-hairpin epitope as novel structural requirement for protein arginine rhamnosylation

For canonical asparagine glycosylation, the primary amino acid sequence that directs glycosylation at specific asparagine residues is well-established. Here we reveal that a recently discovered bacterial enzyme EarP, that transfers rhamnose to a specific arginine residue in its acceptor protein EF-P, specifically recognizes a beta-hairpin loop. Notably, while the in vitro rhamnosyltransferase activity of EarP is abolished when presented with linear substrate peptide sequences derived from EF-P, the enzyme readily glycosylates the same sequence in a cyclized beta-hairpin mimic. Additional studies with other substrate-mimicking cyclic peptides revealed that EarP activity is sensitive to the method used to induce cyclization and in some cases is tolerant to amino acid sequence variation. Using detailed NMR approaches, we established that the active peptide substrates all share some degree of beta-hairpin formation, and therefore conclude that the beta-hairpin epitope is the major determinant of arginine-rhamnosylation by EarP. Our findings add a novel recognition motif to the existing knowledge on substrate specificity of protein glycosylation, and are expected to guide future identifications of rhamnosylation sites in other protein substrates

A Sensitive Gel-Based Method Combining Distinct Cyclophellitol-Based Probes for the Identification of Acid/Base Residues in Human Retaining Beta-Glucosidases

Medical Biochemistr

Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.Bio-organic Synthesi

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide

Bio-organic Synthesi

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.Bio-organic Synthesi

Stabilization of Glucocerebrosidase by Active Site Occupancy

Macromolecular Biochemistr

On the reactivity and selectivity of donor glycosides in glycochemistry and glycobiology

The processes of glycosidic bond formation and destruction are a central theme in glycochemistry and glycobiology, and form the basis of the research described in this Thesis. In the first part, studies towards the stereoselective construction of two complex bacterial oligosaccharide fragments are described. These fragments contain mannuronic acid residues connected through a beta-linkage, which is amongst the most challenging linkages to construct synthetically. In the second part, the use of mannuronic acid building blocks in the automated synthesis of alginates using solid-phase chemistry is presented. Using a second-generation carbohydrate synthesizer, alginate fragments up to 12 residues were assembled with high stereoselectivity. Using the same automated set-up, fragments of hyaluronic acid of up to 15 residues were synthesized. In the third part, 2-deoxy-2-fluoroglucosides are investigated as activity-based probes for glucocerebrosidase, a retaining beta-glucosidase enzyme

A β-hairpin epitope as novel structural requirement for protein arginine rhamnosylation

Microbial Biotechnolog