Semiprocessive Hyperglycosylation of Adhesin by Bacterial Protein N-Glycosyltransferases

[Image: see text] 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.