Structural studies of an A2-type modular polyketide synthase ketoreductase reveal features controlling α-substituent stereochemistry.
J.Zheng,
S.K.Piasecki,
A.T.Keatinge-Clay.
ABSTRACT
Modular polyketide synthase ketoreductases often set two stereocenters when
reducing intermediates in the biosynthesis of a complex polyketide. Here we
report the 2.55-Å resolution structure of an A2-type ketoreductase from the
11th module of the amphotericin polyketide synthase that sets a combination of
l-α-methyl and l-β-hydroxyl stereochemistries and represents the final
catalytically competent ketoreductase type to be structurally elucidated.
Through structure-guided mutagenesis a double mutant of an A1-type ketoreductase
was generated that functions as an A2-type ketoreductase on a diketide substrate
analogue, setting an α-alkyl substituent in an l-orientation rather than in the
d-orientation set by the unmutated ketoreductase. When the activity of the
double mutant was examined in the context of an engineered triketide lactone
synthase, the anticipated triketide lactone was not produced even though the
ketoreductase-containing module still reduced the diketide substrate analogue as
expected. These findings suggest that re-engineered ketoreductases may be
catalytically outcompeted within engineered polyketide synthase assembly lines.
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