Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1
| Autor: | Lubbert Dijkhuizen, Bauke W. Dijkstra, Reinetta M. Buitelaar, Richèle D. Wind, Joost C.M. Uitdehaag |
|---|---|
| Přispěvatelé: | Groningen Biomolecular Sciences and Biotechnology, X-ray Crystallography, Host-Microbe Interactions |
| Jazyk: | angličtina |
| Rok vydání: | 1998 |
| Předmět: |
Models
Molecular alpha-Cyclodextrins Stereochemistry ALPHA-AMYLASE Archaeal Proteins alpha-Cyclodextrin Molecular Conformation Oligosaccharides ANGSTROM RESOLUTION Cyclodextrin glycosyltransferase Crystallography X-Ray Protein Engineering Biochemistry X-RAY STRUCTURE Active center Hydrolysis chemistry.chemical_compound NUCLEOTIDE-SEQUENCE CATALYTIC MECHANISM Enzyme Stability PROTEIN MODELS Glycoside hydrolase Enzyme Inhibitors Molecular Biology chemistry.chemical_classification Cyclodextrins Binding Sites Cyclodextrin Chemistry Starch Cell Biology Protein engineering Hydrogen-Ion Concentration Archaea BACILLUS-CIRCULANS STRAIN-251 Glucosyltransferases ESCHERICHIA-COLI Mutagenesis Site-Directed Bacillus circulans CYCLIZATION CHARACTERISTICS ACTIVE-CENTER Protein Binding |
| Zdroj: | The Journal of Biological Chemistry, 273(10), 5771-5779. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC |
| ISSN: | 0021-9258 |
| Popis: | The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltohexaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-Angstrom resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation, We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase.The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site directed mutagenesis, Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD.Glu(258) is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu(258) in the CGTase from T. thermosulfurigenes EM1 were changed. Phe(284) was replaced by Lys and Asn(327) by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme, This suggests that the pH optimum curve of CGTase is determined only by residue Glu(258). |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|