title: Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution.
abstract: 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.
authors.affiliation: School of Biochemistry and Molecular Biology, Department of Chemistry, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Williams
authors.forename: Silvie
authors.initials: S
authors.lastname: Domann
authors.forename: Adam
authors.initials: A
authors.lastname: Nelson
authors.forename: Alan
authors.initials: A
authors.lastname: Berry
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D005635:Fructosediphosphates / Q000737:chemistry / Q000378:metabolism; D006598:Hexosediphosphates / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D005635:Fructosediphosphates; D006598:Hexosediphosphates; D011994:Recombinant Proteins; C026633:D-tagatose 1,6-diphosphate; D000446:Aldehyde-Lyases; C023093:tagatose 1,6-diphosphate aldolase; C029063:fructose-1,6-diphosphate