Abstract: In order to search for oxidoreductases suitable
for the preparation of methyl (R)-o-chloromandelate
[(R)-CMM], the key intermediate for clopidogrel,
the homologous proteins of Gre2p were expressed
in Escherichia coli, among which CgKR1
showed the most satisfactory activity and stereoselectivity
towards methyl o-chlorobenzoylformate
(CBFM). Using the crude enzyme of CgKR1 and
glucose dehydrogenase (GDH), as much as 300 g·L1
of CBFM was almost stoichiometrically converted to
(R)-CMM with excellent enantiomeric excess (98.7%
ee). More importantly, the reaction could be performed
without external addition of an expensive cofactor.
The substrate profile indicates that keto
esters serve as the most suitable substrate, which was
confirmed by gram-scale preparations. Homology
modeling and docking analysis revealed the molecular
basis for the high stereoselectivity of CgKR1.
These demonstrate not only the feasibility of in silico
mining of novel enzymes based on sequence homology
but also the applicability of this new reductase for
the practical production of optically active (R)-
CMM.