Studying the Role of a Single Mutation of a Family 11 Glycoside Hydrolase Using High-Resolution X-ray Crystallography
| Autor: | Chunran Li, Andrey Kovalevsky, Qun Wan, Zhihong Li, Xiaoshuai Zhang |
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| Rok vydání: | 2020 |
| Předmět: |
Glycoside Hydrolases
Stereochemistry Mutant Mutation Missense Bioengineering Crystallography X-Ray Biochemistry Analytical Chemistry Catalysis Fungal Proteins 03 medical and health sciences Residue (chemistry) Protein Domains Bioorganic chemistry Glycoside hydrolase 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology 030302 biochemistry & molecular biology Organic Chemistry Mutagenesis Active site Enzyme chemistry Hypocreales biology.protein |
| Zdroj: | The Protein Journal. 39:671-680 |
| ISSN: | 1875-8355 1572-3887 |
| Popis: | 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 pKa calculations show that D44 has similar position and pKa 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. |
| Databáze: | OpenAIRE |
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