American Chemical Society
cs5b02805_si_001.pdf (2.27 MB)

Minimalist Protein Engineering of an Aldolase Provokes Unprecedented Substrate Promiscuity

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journal contribution
posted on 2016-02-04, 00:00 authored by Deniz Güclü, Anna Szekrenyi, Xavier Garrabou, Michael Kickstein, Sebastian Junker, Pere Clapés, Wolf-Dieter Fessner
Application of aldolases for the asymmetric synthesis of multifunctional chiral products is hampered by their reputed strict nucleophile (=aldol donor) specificity owing to a mechanistic requirement for creating a carbanion nucleophile in aqueous medium. Here we report that a minimalist engineering can extensively broaden the substrate scope of native d-fructose-6-phosphate aldolase (FSA) from Escherichia coli, for which hydroxyacetone is the most proficient substrate, to accept an unprecedented wide variety of alternative nucleophiles. By single- or double-space-generating mutations using simple conservative Leu to Ala replacement of active site residues, we found enzyme variants to efficiently convert larger ketols and bioisosteric ether components with up to seven skeletal atoms, including linear and branched-chain structures. All reactions occurred with full retention of the natural d-threo diastereospecificity. These FSA variants open new avenues toward the synthesis of novel product families that hitherto were inaccessible by biological catalysis.