The Transaldolase Family: New Synthetic Opportunities from an Ancient Enzyme Scaffold

Abstract

Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalytic carboligation by aldolases offers a green, uniquely regio- and stereoselective tool with which to perform these transformations. Recent advances in the field, fueled by both discovery and protein engineering, have greatly improved the synthetic opportunities for the atom-economic asymmetric synthesis of chiral molecules with potential pharmaceutical relevance. New aldolases derived from the transaldolase scaffold (based on transaldolase B and fructose-6-phosphate aldolase from Escherichia coli) have been shown to be unusually flexible in their substrate scope; this makes them particularly valuable for addressing an expanded molecular range of complex polyfunctional targets. Extensive knowledge arising from structural and molecular biochemical studies makes it possible to address the remaining limitations of the methodology by engineering tailored biocatalysts.

Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions

“…Remarkably, a single variant can be engineered to produce opposite selectivity at each aldol step, as in the synthesis of 6-chloro-L-glucose (5j). Such active site malleability demonstrates the great evolutionary potential for the functional diversification of FSA, which nature has not exploited, as inferred from the reduced sequence variability observed within the FSA family 23,40 . Application of higher-throughput screening techniques can be expected to boost the fast evolution of FSA and its variants towards higher activity and stereoselectivity, and enable unprecedented non-biological reactivity.…”

Fluorescence‐Based Kinetic Assay for High‐Throughput Discovery and Engineering of Stereoselective ω‐Transaminases

“…[23] Each enzyme-substrate pair was measured in duplicate using as erial 2 dilution from 10 to 0. 16 Thea ssay was evaluated using the w-transaminases from Cv (0.66 U/mg), Vf (15.8 U/mg), AspFum (0.019 U/mg), AspTer (0.00078 U/mg), NeoFis (0.013 U/mg) and Codexis enzymes ATA113 (0.45 U/mg) and ATA117 (1.9 U/mg). Costar 96 well plates were used in aB MG Clariostar plate reader with 330 nm excitation and 460 nm emission set for detection.A ll stock solutions were prepared with HEPES buffer (50 mM, pH 8.2).…”

Enzyme‐Catalyzed Aldol Additions

“…This single amino acid substitution changed the enzyme from aldol transfer to a freely dissociating aldol activity and led to a 70-fold increase in the V max for the formation of d-fructose-6-phosphate well into the range of FSA activity. The reader can find more comprehensive information concerning the biological function, structural and biochemical features, and synthetic applications of the transaldolase family in a recently published review [12].…”

“…In this chapter, we summarize the major progress of the last years. For a more comprehensive coverage of the general topic, we refer the readers to previous reviews [12][13][14][15][16][17][18][19][20][21][22][23].…”