Directed Evolution of a Glycosynthase via Chemical Complementation
- 1 November 2004
- journal article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 126 (46) , 15051-15059
- https://doi.org/10.1021/ja046238v
Abstract
Recently, we reported a general assay for enzyme catalysis based on the yeast three-hybrid assay, Chemical Complementation, which is intended to expand the range of chemical reactions to which directed evolution can be applied. Here, Chemical Complementation was applied to a glycosynthase derived from a retaining glycosidase, an important class of enzymes for carbohydrate synthesis. Using the yeast three-hybrid assay, the glycosynthase activity of the E197A mutant of the Cel7B from Humicola insolens was linked to transcription of a LEU2 reporter gene, making cell growth dependent on glycosynthase activity in the absence of leucine. Then the LEU2 selection was used to isolate the most active glycosynthase from a Glu197 saturation library, yielding an E197S Cel7B variant with a 5-fold increase in glycosynthase activity. These results not only establish Chemical Complementation as a platform for the directed evolution of glycosynthases, but also show the generality of this approach and the ease with which it can be applied to new chemical reactions.Keywords
This publication has 35 references indexed in Scilit:
- Mutated Barley (1,3)-β-d -Glucan Endohydrolases Synthesize Crystalline (1,3)-β-d -GlucansJournal of Biological Chemistry, 2002
- A Bacterial Small-Molecule Three-Hybrid SystemAngewandte Chemie International Edition in English, 2002
- The Enzymatic Synthesis of Glycosidic Bonds: "Glycosynthases" and Glycosyltransferases.Trends in Glycoscience and Glycotechnology, 2001
- Chemical and enzymatic synthesis of glycopolymersCurrent Opinion in Chemical Biology, 2000
- Highly Efficient Synthesis of β(1 → 4)-Oligo- and -Polysaccharides Using a Mutant CellulaseJournal of the American Chemical Society, 2000
- The E358S mutant of Agrobacterium sp. β‐glucosidase is a greatly improved glycosynthaseFEBS Letters, 2000
- From β‐glucanase to β‐glucansynthase: glycosyl transfer to α‐glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophileFEBS Letters, 1998
- Structure of the Fusarium oxysporum Endoglucanase I with a Nonhydrolyzable Substrate Analogue: Substrate Distortion Gives Rise to the Preferred Axial Orientation for the Leaving Group,Biochemistry, 1996
- Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulasesEuropean Journal of Biochemistry, 1993
- N-Sulfinylnonafluorobutanesulfonamide—A SupernucleophileAngewandte Chemie International Edition in English, 1980