Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase

d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimThis project has received funding from the Auvergne council, the Bundesministerium für Bildung und Forschung (BMBF grant 0315775B PT-J to W.-D.F.), Ministerio de Economía y Competitividad (MINECO) (grant CTQ2015-63563-R to P.C. and CTQ2015-64436-P to T.P.) and COST action CM1303 Systems Biocatalysis. Anne Samland (Stuttgart University) is warmly thanked for a gift of plasmid for expressing FSA K85M.Peer reviewe