Minimalist Protein Engineering of an Aldolase Provokes Unprecedented Substrate Promiscuity

Abstract

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.

Nucleophile Promiscuity of Natural and Engineered Aldolases

“…Upon increasing steric space in the hydrophobic nucleophile‐binding domain of FSA (i.e., residues L107 and L163), by using simple conservative Leu to Ala replacement, FSA L107A/L163A , the nucleophile repertoire of FSA was largely expanded . Hence, higher ketols; analogues of 1 b ; and bioisosteric ethers, which are analogues of 1 a , with up to seven skeletal atoms, including linear and branched‐chain structures were efficiently converted (Scheme ).…”

“…L163A was the critical mutation that created the additional space necessary to accommodate larger substrates in a rather extended conformation, whereas the L107A/L163A variant, which offered the contiguous extra void, now enabled the binding of ketols with chain lengths (at least) of up to C7 carbon atoms …”

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

“…Remarkably, it accepts hydroxyethanal (HE) as an aldehyde rather than a ketone nucleophile (Figure ), which is unparalleled among the aldolases, with the exception of 2‐deoxy‐ d ‐ribose‐5‐phosphate aldolase (DERA), which uses ethanal. More recently, the subtle nucleophile selectivity of wild‐type FSA was dramatically enhanced by minimalist protein engineering . Single or double active site mutations allowed an efficient conversion of larger ketol components with up to seven skeletal atoms.…”

Recent Developments in the Preparation of Carbohydrate Derivatives from Achiral Building Blocks by using Aldolases

“…In a very recent contribution, the same research group demonstrated that the substrate scope of FSA can be spectacularly broadened with a minimum of active site engineering . Optimal results were obtained with the double variant FSA L107A L 163A in which bulky Leu at the active site have been replaced by less hindered Ala.…”