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Enzymatic hydroxylation of activated and nonactivated sp3-carbons
attracts keen interest from the chemistry community as it is one of the
most challenging tasks in organic synthesis. Nature provides a vast
number of enzymes with an enormous catalytic versatility to fulfill this
task. Given that those very different enzymes have a distinct
specificity in substrate scope, selectivity, activity, stability, and
catalytic cycle, it is interesting to outline similarities and
differences. In this Review, we intend to delineate which enzymes
possess considerable advantages within specific issues. Heterologous
production, crystal structure availability, enzyme engineering
potential, and substrate promiscuity are essential factors for the
applicability of these biocatalysts.
Publikation
Knorrscheidt, A.; Soler, J.; Hünecke, N.; Püllmann, P.; Garcia-Borràs, M.; Weissenborn, M. J.;Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific PeroxygenaseACS Catal.117327-7338(2021)DOI: 10.1021/acscatal.1c00847
Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad
substrate range with unparalleled activities. Tailoring these enzymes
for chemo- and regioselective transformations represents a grand
challenge due to the difficulties in their heterologous productions.
Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila.
More than 5300 transformants were screened. This protein engineering
led to a significant reshaping of the active site as elucidated by
computational modelling. The reshaping was responsible for the increased
oxyfunctionalization activity, with improved kcat/Km
values of up to 16.5-fold for the model substrate
5-nitro-1,3-benzodioxole. Moreover, variants were identified with high
chemo- and regioselectivities in the oxyfunctionalization of aromatic
and benzylic carbons, respectively. The benzylic hydroxylation was
demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO
variants represent a step forward toward the use and implementation of
UPOs in biocatalytic synthetic pathways of industrial interest.