Angewandte Chemie International Edition
10.1002/anie.202012675
RESEARCH ARTICLE
screened employing a high-throughput colorimetric UDCA assay.
Starting from an enzyme which did not even have trace activity in
the formation of the target compound, we were able to create a
variant that produces mainly UDCA, with only traces of MDCA
formed. The near complete inversion of regioselectivity from 6β-
(
MDCA formation) to 7β-hydroxylation (UDCA production)
demonstrates how protein engineering can be used to create
custom biocatalysts, even for reactions where no naturally
occurring counterpart has yet been identified.
Acknowledgements
We thank the Technologie-Beratungs-Institut GmbH for funding of
the P450-OH project (grant number: TBI-V-1-198-VBW-068). We
are also grateful to Dr. Henk-Jan Joosten for useful discussions
related to the 3DM database used. T.B. was supported by the
Austrian Science Fund (FWF) through the Erwin Schrödinger
Fellowship (grant number: J4231-B21). S.W. was supported by a
Humboldt research fellowship.
Figure 2. UDCA-producing variants with increased selectivity. OleP variants
show increasing selectivity towards UDCA (blue) and increased UDCA
formation (red). UDCA was quantified by HPLC of three replicate reactions.
Conflict of interest
S.K. and H.B. are employees of Enzymicals AG and B.G. is an
employee of HERBRAND PharmaChemicals GmbH.
To get further insights into the contribution of each position and to
find the most selective variant, we created a set of 37 single,
double, and triple mutants at residues F84, S240A, and V291.
This selection was based on UDCA:MDCA ratios of the individual
variants and sequencing of UDCA producing variants (Figure S6
and Table S3). All newly created variants were analyzed by the
colorimetric assay as well as HPLC. Several single, double, and
triple mutants (Figure 2) were capable of producing UDCA
Keywords: Ursodeoxycholic acid • 7β-hydroxylation • lithocholic
acid • protein engineering • cytochrome P450 monooxygenase
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