Directed evolution of transaldolases for novel specificities
Final Report Abstract
Aldolases are powerful catalysts for asymmetric synthesis in that they control the creation of up to two new stereo-centers, yet their natural substrate specificities and stereo-selectivities are limiting. We aimed to produce modified transaldolases with new donor substrate specificities and unnatural stereo-specificities by directed evolution, guided by information from protein structures of natural transaldolases and fructose 6-phosphate aldolase, FSA. Because of mechanistic considerations, such protein modifications involved active-site mutations that usually would be deleterious to catalytic activity. For diversity generation, we used focused saturation mutagenesis at eleven residues of the E. coli enzyme, transaldolase B (TalB). Combined with a newly developed efficient screening assay, we were able to select a single enzyme variant (Phe178Tyr, F178Y) which displayed an enormously improved activity for the donor compound, dihydroxyacetone matching almost the activity of fructose-6-phosphate aldolase, FSA. The 3D structure of the F178Y variant was solved and showed that the hydroxyl group of Tyr178 residue plays an important role in this gain-of-function mutation. This 3D structure also hinted us to a possible binding site for the phosphate moiety of sugar phosphates at TalB´s active site. In a second round of saturation mutagenesis, we were able to select enzyme variants of TalB F178Y which had improved activities for unphosphorylated acceptor compounds such as glyceraldehyde, delivering fructose as product. Our aim to obtain TalB variant with altered stereoselectivities, however, has not been successful so far.
