α,α′-Dihydroxyketone formation using aromatic and heteroaromatic aldehydes with evolved transketolase enzymes

Abstract

Transketolase mutants have been identified that accept aromatic acceptors with good stereoselectivities, in particular benzaldehyde for which the wild type enzyme showed no activity.

Structural stability of E. coli transketolase to temperature and pH denaturation

“…(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) coli transketolase by directed evolution has increased the enzyme activity , modified the substrate specificity to better accept aliphatic aldehydes , and both enhanced and reversed the enantioselectivity with aliphatic and (hetero)aromatic aldehyde substrates (Cazares et al, 2010;Galman et al, 2010;Smith et al, 2008). Such exquisite stereochemical control coupled to carbon-carbon bond formation will be particularly important for meeting the increasing demand for enantiopure pharmaceuticals Koeller and Wong, 2001).…”

Directed evolution to re-adapt a co-evolved network within an enzyme

“…Various single mutants gave improved activity towards glycolaldehyde (GA) (Hibbert et al, 2007), or accepted propionaldehyde (PA) better with either enhanced or reversed enantioselectivity (Hibbert et al, 2008; Smith et al, 2008; Hailes et al, 2010). Several D469 mutations also accepted a range of linear aliphatic (Cazares et al, 2010) and aromatic (Galman et al, 2010) aldehydes. Chemical denaturation, dimer interface mutations and biocatalytic process conditions that destabilise Escherichia coli TK have also been well characterised (Brocklebank et al, 1999; Martinez-Torres et al, 2007; Aucamp et al, 2008).…”

Second generation engineering of transketolase for polar aromatic aldehyde substrates

“…The structures of TK variants, aldehydes and ThDP enamine were modelled as previously described [33] (see SI for further details) into TK variant structures prepared by replacing the target residues of the E. coli wild type (PDB ID: 1QGD) [46]. We define "productive location/proximity" in all cases simply as placing the aldehyde carbonyl and the enamine-cofactor intermediate at within ca.…”

“…Double-reciprocal Lineweaver-Burk plots were also used to verify that the relationships between the velocity and the concentration were linear. The racemic products from 3-HBA [33], 3-FBA and 4-FBA [37] were synthesised, purified, and characterised as previously, as standards for HPLC calibration. None of the aromatic product enantiomers could be directly resolved by chiral-HPLC and so derivatisation was attempted using several methods.…”

“…Indeed, protein engineering of WT E. coli TK has been successfully used to improve its activity towards aliphatic aldehydes [31], with both enhanced and reversed stereospecificity [32][33][34][35], and also improved activity towards d-ribose and d-glucose [36]. By contrast, aromatic aldehydes still suffer from low reaction yields [33], except 3-formylbenzoic acid (3-FBA) and 4-formylbenzoic acid (4-FBA) which introduce a beneficial binding interaction between their carboxylic acid group and the transketolase phosphate-binding residues [37]. The E. coli TK variant D469T was found previously to have the highest activity towards the carboxylated aromatic substrates 3-FBA and 4-FBA, from all TK variants tested [37].…”