Supplementary document for Ultracompact Vernier-effect-improved sensor by a single microfiber-knot resonator - 6263535.pdf

supplement materia

Supplementary document for Ultracompact Vernier-effect-improved sensor by a single microfiber-knot resonator - 6237197.pdf

supplemental material

Understanding the pH-Dependent Reaction Mechanism of a Glycoside Hydrolase Using High-Resolution X‑ray and Neutron Crystallography

Glycoside hydrolases (GHs) commonly use the retaining or inverting mechanisms to hydrolyze carbohydrates, and the rates of catalysis are usually pH dependent. Deeper understanding of these pH-dependent reaction mechanisms is of great importance for protein engineering and drug design. We used high-resolution X-ray crystallography to analyze the sugar ring configurations of an oligosaccharide ligand during hydrolysis for the family 11 GH, and the results support the ¹S₃ → ⁴H₃ → ⁴C₁ conformational itinerary. These results indicate that sugar ring flexibility may help to distort and break the glycosidic bond. Constant pH molecular dynamics simulations and neutron crystallography demonstrate that the catalytic glutamate residue (E177) has alternate conformational changes to transfer a proton to cleave the glycosidic bond. Furthermore, a neutron crystallography analysis shows that the H-bond length between E177 and its nearby tyrosine residue (Y88) is shortened when the pH increases, preventing E177 from rotating downward and obtaining a proton from the solvent for catalysis. This result indicates that the H-bond length variation may play a key role in the pH-dependent reaction mechanism. In summary, our results demonstrate that both sugar ring flexibility and protein dynamics are important in the pH-dependent reaction mechanism and may help to engineer GHs with different pH optima