Minimalist Protein Engineering of an Aldolase Provokes
Unprecedented Substrate Promiscuity
Version 2 2016-02-29, 17:20
Version 1 2016-02-17, 14:48
Posted on 2016-02-29 - 17:19
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.
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Güclü, Deniz; Szekrenyi, Anna; Garrabou, Xavier; Kickstein, Michael; Junker, Sebastian; Clapés, Pere; et al. (2016). Minimalist Protein Engineering of an Aldolase Provokes
Unprecedented Substrate Promiscuity. ACS Publications. Collection. https://doi.org/10.1021/acscatal.5b02805
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AUTHORS (7)
DG
Deniz Güclü
AS
Anna Szekrenyi
XG
Xavier Garrabou
MK
Michael Kickstein
SJ
Sebastian Junker
PC
Pere Clapés
WF
Wolf-Dieter Fessner
KEYWORDS
Aldolase Provokes Unprecedented Substrate PromiscuityApplicationAla replacementMinimalist Protein Engineeringmultifunctional chiral productssubstrate scopecarbanion nucleophilealternative nucleophilessynthesisaldolasesite residuesnovel product familiesbioisosteric ether componentsEscherichia colienzyme variantsFSA variants