Williams, G.J, Domann, S., Nelson, A. et al. (1 more author) (2003) Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution. Proceedings of the National Academy of Sciences, 100 (6). pp. 3143-3148. ISSN 0027-8424
Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates. Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase. After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k-cat/K-m toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity. (31)P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon—carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate. This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics.
|Copyright, Publisher and Additional Information:||© Copyright 2003 National Academy of Sciences, U.S.A.|
|Institution:||The University of Leeds|
|Academic Units:||The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds)|
|Depositing User:||Sherpa Assistant|
|Date Deposited:||13 Mar 2006|
|Last Modified:||25 Oct 2016 14:01|