Biomedical subjects

Haiyan Tao

Publications and source records attributed to Haiyan Tao.

3 recordsLinked to original sources

PubMed2006

Optimized design and synthesis of chemical dimerizer substrates for detection of glycosynthase activity via chemical complementation.

Glycosynthases catalyze the formation of a glycosidic bond between a glycosyl fluoride donor substrate and a glycosyl acceptor substrate with high yield, thus providing a valuable approach for the synthesis of carbohydrates and glycoconjugates. Chemical complementation can be used to link glycosynthase activity to the transcription of a reporter gene in vivo, providing a selection for the directed evolution of glycosynthase enzymes with improved properties. In this approach, glycosynthase activity is detected as covalent coupling between a small molecule disaccharide acceptor substrate and a small molecule disaccharide alpha-fluoro donor substrate. Here we report the optimized design and synthesis of these small molecule substrates. These optimized substrates are shown to give a robust, glycosynthase-dependent transcriptional read-out in the chemical complementation assay. The full synthesis and characterization of these substrates are reported for the first time. These optimized chemical dimerizer substrates should allow the potential of chemical complementation for the directed evolution of glycosynthases with diverse substrate specificities and improved properties to be fully realized.

Binding Sites↗

PubMed2004

Directed evolution of a glycosynthase via chemical complementation.

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.

Ascomycota↗

PubMed2002

Milestones in directed enzyme evolution.

Directed evolution has now been used for over two decades as an alternative to rational design for protein engineering. Protein function, however, is complex, and modifying enzyme activity is a tall order. We can now improve existing enzyme activity, change enzyme selectivity and evolve function de novo using directed evolution. Although directed evolution is now used routinely to improve existing enzyme activity, there are still only a handful of examples where substrate selectivity has been modified sufficiently for practical application, and the de novo evolution of function largely eludes us.

Catalysis↗