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an approx. 1:1 mixture of the expected cis-5-hydroxypipecolic
acid and its cis-3-isomer, which is also the product of cis-
P3H_II, was obtained [34]. In general, it can be assumed that
the shift of reactivity in the reaction with trans-3-methyl-L-
proline is due to the increased stability of the tertiary radical
intermediate at C-3 compared to the secondary at one C-4.
However, this putative effect does not increase the reactivity of
cis-P3H_II. So it is most likely that the complex interplay
between kinetic and steric factors determines the reactivity of
these enzymes. Further spectroscopic and structural data are
required in order to provide an insight into the functionality of
these enzymes. Nevertheless, our results show that α-KG
dependent oxygenases have high potential for the production of
tertiary alcohols. Both enzymes investigated afford only a
single product selectively and, in the case of cis-P4H, the
activity was comparable to that with the native substrate. More-
over, proline hydroxylases can be applied for whole cell
biotransformations on a preparative scale. Even though the
activity of the enzymes is still difficult to predict for conver-
sions with unnatural substrates, highly efficient catalytic
systems may be accessible from other α-KG dependent oxyge-
nases.
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We thank Prof. Michael Müller for his generous support. The
skillful technical assistance of Olga Fuchs and Alexandra
Walter is gratefully acknowledged. We also thank Volker
Brecht for measuring the NMR spectra. This work was finan-
cially supported by the Deutsche Bundesstiftung Umwelt
(DBU, ChemBioTec: AZ13234-32).
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