Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution
article
Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common 'intermediate-like' ancestor towards specific transglycosylation activity. © The Authors.
Chemicals/CAS: alpha cyclodextrin, 10016-20-3; beta cyclodextrin, 7585-39-9; cyclomaltodextrin glucanotransferase, 9030-09-5; gamma cyclodextrin, 17465-86-0; glycosidase, 9032-92-2; cyclomaltodextrin glucanotransferase, EC 2.4.1.19; Glucosyltransferases, EC 2.4.1.-
Chemicals/CAS: alpha cyclodextrin, 10016-20-3; beta cyclodextrin, 7585-39-9; cyclomaltodextrin glucanotransferase, 9030-09-5; gamma cyclodextrin, 17465-86-0; glycosidase, 9032-92-2; cyclomaltodextrin glucanotransferase, EC 2.4.1.19; Glucosyltransferases, EC 2.4.1.-
Topics
AmylaseCyclodextrin glucanotransferase (CGTase)Protein evolutionProtein stabilitySaccharideSide reactionAminesCrystal structureCyclizationCyclodextrinsEnzyme activityEnzymesFood additivesHydrolysisIntegral equationsPlants (botany)SilanesSugar (sucrose)SugarsActive sitesAnalysis of variousBreakdown (BD)Catalytic functionsCatalytic sitesConformational changesCyclization activityCyclodextrin (CyDs)Cyclodextrin glucanotransferase (CGTase)Directed evolutionEnzyme functioningError prone PCRGlycoside hydrolases (GH)Hydrolysis reactionsHydrolytic activitiesPeptide backbonesSide chainsSide reactionsTrans glycosylation activityRate constantsCalorimetry, Differential ScanningCatalysisChromatography, High Pressure LiquidEvolution, MolecularGlucosyltransferasesHydrolysisModels, MolecularMutagenesisPolymerase Chain ReactionProtein Structure, SecondaryThermoanaerobacterium thermosulfurigenes
TNO Identifier
280037
ISSN
0264-6021
Source
Biochemical Journal, 413(3), pp. 517-525.
Pages
517-525
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