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Processivity is an important feature of enzyme families such as DNA polymerases, polysaccharide synthases, and protein kinases, to ensure high fidelity in biopolymer synthesis and modification. Here, we reveal processive character in the family of cytoplasmic protein N-glycosyltransferases (NGTs). Through various activity assays, intact protein mass spectrometry, and proteomics analysis, we established that NGTs from nontypeable Haemophilus influenzae and Actinobacillus pleuropneumoniae modify an adhesin protein fragment in a semiprocessive manner. Molecular modeling studies suggest that the processivity arises from the shallow substrate binding groove in NGT, which promotes the sliding of the adhesin over the surface to allow further glycosylations without temporary dissociation. We hypothesize that the processive character of these bacterial protein glycosyltransferases is the mechanism to ensure multisite glycosylation of adhesins in vivo, thereby creating the densely glycosylated proteins necessary for bacterial self-aggregation and adherence to human cells, as a first step toward infection.

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 β-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 β-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 β-hairpin formation, and therefore conclude that the β-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., For bacterial arginine rhamnosylation, the rhamnosyltransferase EarP specifically recognizes a β-hairpin structure in the acceptor substrate.

Extracellular polysaccharides and glycoproteins of pathogenic bacteria assist in adherence, autoaggregation, biofilm formation, and host immune system evasion. As a result, considerable research in the field of glycobiology is dedicated to study the composition and function of glycans associated with virulence, as well as the enzymes involved in their biosynthesis with the aim to identify novel antibiotic targets. Especially, insights into the enzyme mechanism, substrate binding, and transition-state structures are valuable as a starting point for rational inhibitor design. An intriguing aspect of enzymes that generate or process polysaccharides and glycoproteins is the level of processivity. The existence of enzymatic processivity reflects the need for regulation of the final glycan/glycoprotein length and structure, depending on the role they perform. In this Review, we describe the currently reported examples of various processive enzymes involved in polymerization and transfer of sugar moieties, predominantly in bacterial pathogens, with a focus on the biochemical methods, to showcase the importance of studying processivity for understanding the mechanism.

Sugars are fascinating and highly diverse molecules with a myriad of roles in all living cells. Importantly, bacteria often utilize sugars in their infection strategies. They are found on the surface of bacterial cells as parts of the larger structures responsible for movement, protection, adhesion, camouflage and interactions with the host immune system. In addition, sugars also decorate the biomolecules inside the cell, influencing their properties and functions, keeping the bacterial cell running. Interestingly, bacterial sugars are frequently distinctly different from human, which means that we can potentially target infectious bacteria using these sugars without damaging our own systems. In the projects described in this thesis, the production systems of important sugar structures from infectious bacteria were investigated and their mechanism studied in detail. For example, in one project, it was elucidated how surface adhesion molecules are decorated with multiple sugars and how to manipulate this process. In another project it was uncovered why only specific proteins are decorated with a rare bacterial sugar. With these findings our understanding of bacterial sugar systems and their roles in infection was expanded. Additionally, by uncovering parts of the mechanism, useful knowledge was gained for developing molecules that can efficiently and selectively block these processes.

Processivity is an important feature of enzyme families such as DNA polymerases, polysaccharide synthases and protein kinases, to ensure high fidelity in biopolymer synthesis and modification. Here we reveal processive character in the family of cytoplasmic protein N-glycosyltransferases (NGTs). Through various activity assays, intact protein mass spectrometry and proteomics analysis, we established that NGTs from non-typeable Haemophilus influenzae and Actinobacillus pleuropneumoniae modify an adhesin protein fragment in a semi-processive manner. Molecular modeling studies suggest that the processivity arises from the shallow substrate binding groove in NGT, that promotes the sliding of the adhesin over the surface to allow further glycosylations without temporary dissociation. We hypothesize that the processive character of these bacterial protein glycosyltransferases is the mechanism to ensure multisite glycosylation of adhesins in vivo, thereby creating the densely glycosylated proteins necessary for bacterial self-aggregation and adherence to human cells, as a first step towards infection.

With the increasing resistance of bacteria to current antibiotics, novel compounds are urgently needed to treat bacterial infections. Streptozotocin (STZ) is a natural product that has broad-spectrum antibiotic activity, albeit with limited use because of its toxicity to pancreatic &beta, cells. In an attempt to derivatize STZ through structural modification at the C3 position, we performed the synthesis of three novel STZ analogues by making use of our recently developed regioselective oxidation protocol. Keto-STZ (2) shows the highest inhibition of bacterial growth (minimum inhibitory concentration (MIC) and viability assays), but is also the most cytotoxic compound. Pre-sensitizing the bacteria with GlcNAc increased the antimicrobial effect, but did not result in complete killing. Interestingly, allo-STZ (3) revealed moderate concentration-dependent antimicrobial activity and no cytotoxicity towards &beta, cells, and deoxy-STZ (4) showed no activity at all.