Liam Mihalynuk

Topic

Structural and functional characterization of two bacterial O-glycopeptidases reveals sophisticated mechanisms of substrate recognition

School of Molecular Life Sciences

Date & location

• Monday, October 26, 2026
• 1:00 P.M.
• Clearihue Building, Room B017

Examining Committee

Supervisory Committee

• Dr. Alisdair Boraston, School of Molecular Life Sciences, University of Victoria (Supervisor)
• Dr. John Burke, School of Molecular Life Sciences, UVic (Member)
• Dr. Jürgen Ehlting, Department of Biology, UVic (Outside Member)

External Examiner

• Dr. Stephen Tuffs, School of Molecular Life Sciences, UVic

Chair of Oral Examination

• Dr. Chenxi Liu, Department of Computer Science, UVic

Abstract

Host-adapted microorganisms employ diverse enzyme repertoires to degrade mucins, facilitating nutrient acquisition and, in pathogenic contexts, penetration of protective mucus layers. Among these enzymes, O-glycopeptidases are specialized proteases that cleave adjacent to O-glycosylated residues in a glycan-dependent fashion and have emerged as valuable tools for analyzing O-glycosylated proteins. Despite the growing number of predicted O-glycopeptidases, relatively few have been structurally and functionally characterized, leaving the molecular determinants governing their substrate selectivity poorly understood. This thesis investigates substrate recognition by SmE from Serratia marcescens and PvM60 from Phocaeicola vulgatus, the only identifiable O-glycopeptidases encoded by their respective organisms. SmE displayed broad glycan tolerance with a modest preference for 3-sialyl Core 1. The first crystal structures of SmE were determined in apo and glyco-amino-acid-bound forms, revealing the structural basis for its broad glycan tolerance and providing direct insight into glycan recognition by both the catalytic domain and an appended carbohydrate-binding module (CBM). Comparison of eight independently observed SmE conformations further revealed substantial flexibility of the CBM relative to the catalytic domain. Motivated by these observations, novel bis-glycosylated FRET substrates were developed to investigate the contribution of CBM-mediated multivalent substrate recognition to catalysis. Appropriately spaced substrates increased catalytic efficiency by approximately an order of magnitude, while a structure-guided CBM mutation substantially diminished this enhancement, providing direct evidence that the appended CBM potentiates catalysis through multivalent substrate recognition. In contrast, PvM60 exhibited narrow glycan selectivity, with activity restricted to linear glycans and the highest catalytic efficiency toward minimally elaborated glycans. A high-resolution structure and biochemical characterization of its CBM32-like domain confirmed its carbohydrate-binding functionality. However, unlike the SmE CBM, disruption of its carbohydrate-binding site did not reduce catalytic efficiency, suggesting distinct functional roles for the appended CBMs of these enzymes. Together, these findings reveal distinct strategies of substrate recognition and demonstrate how catalytic-site architecture, accessory carbohydrate-binding domains, and the spatial organization of glycosylated substrates collectively shape O-glycopeptidase function.