Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations

Abstract

Chiral auxiliary methodology and chiral assays have been developed to establish the enantiomeric purities of erythrulose and 1,3-dihydroxypentan-2-one generated using wild-type (WT) Escherichia coli transketolase (TK). l-Erythrulose was formed in 95% ee and (3S)-1,3-dihydroxypentan-2-one in 58% ee. Since the latter compound was formed in moderate ee, TK libraries were screened to identify higher performing mutants. A colorimetric screen and chiral assay were successfully applied to a 96-well format, and new active TK mutants were identified, which gave 1,3-dihydroxypentan-2-one in high stereoselectivities. Remarkably, activesite single-point mutants were identified that were able to both enhance and reverse the stereoselectivity of TK.

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).…”

α, α -Dihydroxy Ketones and 2-Amino-1,3-diols: Synthetic and Process Strategies Using Biocatalysts

“…Site directed mutants at these sites had only thus far been shown to be detrimental to activity towards the small -hydroxylated aldehydes or formaldehyde, notably D469A in yeast [51] and D469E in E. coli TK [52]. A third screen of only the D469X and H26X libraries using a chiral GC assay revealed two further mutants H26Y and D469E that produced 1,3-dihydroxypentan-2-one (4) from propanal with 88% ee (3R-isomer) and 90% ee (3S-isomer) respectively (Scheme (5)) [36]. Detailed analysis also of the wild-type, and the D469T and D469Y mutants from the previous screen gave (4) in 58% ee (3S-isomer), 64% ee (3S-isomer), and 53% ee (3R-isomer), respectively.…”

“…Crucial to the success of this assay was the removal of Li-HPA, which also possesses a hydroxy ketone group, using a quaternary amine resin. A protocol was then devised to enable operation in 96-well plates that could rapidly be used to identify active TKs against non--hydroxylated substrates [35,36]. In more recent work the assay has been modified to obtain kinetic data from TK bioconversions [37].…”

Stereoselectivity of an ω-transaminase-mediated amination of 1,3-dihydroxy-1-phenylpropane-2-one

“…Dry THF was obtained using anhydrous alumina columns. 24 All moisture-sensitive reactions were performed under a nitrogen or argon atmosphere using oven-dried glassware. Reactions were monitored by TLC on Kieselgel 60 F 254 plates with detection by UV, potassium permanganate and phosphomolybdic acid stains.…”