Download MendeleyStudying the Role of a Single Mutation of a Family 11 Glycoside Hydrolase Using High-Resolution X-ray Crystallography.
Li, Z., Zhang, X., Li, C., Kovalevsky, A., Wan, Q.(2020) Protein J 39: 671-680
- PubMed: 33128114
- DOI: https://doi.org/10.1007/s10930-020-09938-5
- Primary Citation of Related Structures:
6JUG, 6JWB, 6K9O, 6K9R, 6K9W, 6KW9, 6KWC, 6KWD, 6KWF, 6KWG - PubMed Abstract:
XynII is a family 11 glycoside hydrolase that uses the retaining mechanism for catalysis. In the active site, E177 works as the acid/base and E86 works as the nucleophile. Mutating an uncharged residue (N44) to an acidic residue (D) near E177 decreases the enzyme's optimal pH by ~ 1.0 unit. D44 was previously suggested to be a second proton carrier for catalysis. To test this hypothesis, we abolished the activity of E177 by mutating it to be Q, and mutated N44 to be D or E. These double mutants have dramatically decreased activities. Our high-resolution crystallographic structures and the microscopic pK a calculations show that D44 has similar position and pK a value during catalysis, indicating that D44 changes electrostatics around E177, which makes it prone to rotate as the acid/base in acidic conditions, thus decreases the pH optimum. Our results could be helpful to design enzymes with different pH optimum.
Organizational Affiliation:
College of Science, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
