title: Engineering the donor selectivity of D-fructose-6-phosphate aldolase for biocatalytic asymmetric cross-aldol additions of glycolaldehyde.
abstract: D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity.
authors.affiliation: Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Cataluña (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona (Spain), Fax: (+34) 932045904.
authors.forename: Anna
authors.initials: A
authors.lastname: Szekrenyi
authors.forename: Anna
authors.initials: A
authors.lastname: Soler
authors.forename: Xavier
authors.initials: X
authors.lastname: Garrabou
authors.forename: Christine
authors.initials: C
authors.lastname: Guérard-Hélaine
authors.forename: Teodor
authors.initials: T
authors.lastname: Parella
authors.forename: Jesús
authors.initials: J
authors.lastname: Joglar
authors.forename: Marielle
authors.initials: M
authors.lastname: Lemaire
authors.forename: Jordi
authors.initials: J
authors.lastname: Bujons
authors.forename: Pere
authors.initials: P
authors.lastname: Clapés
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000235:genetics* / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods
chemical_list: D000447:Aldehydes; D029968:Escherichia coli Proteins; D005636:Fructosephosphates; C027618:fructose-6-phosphate; C116609:3-hydroxybutanal; D000446:Aldehyde-Lyases; C426024:fsaA protein, E coli; D000079:Acetaldehyde; C010972:glycolaldehyde
keywords: aldol reaction; carbohydrates; enzyme catalysis; enzyme models; protein engineering