Angewandte
Communications
Chemie
To test this hypothesis further, we generated the variants
IPR E238Y and MNMR Y244E and performed biotransfor-
mation reactions to detect ketoreduction and/or double bond
reduction (Table 2). We tested IPR E238Y at pH 6.0, consis-
tent with the preference for lower pH values of the wild-type
enzymes, in addition to reactions at pH 7.0 for comparison
with the MNMR Y244E variant. IPR E238Y showed no
double bond reduction with any substrate tested (3a,b and
fructose-6-phosphate aldolase) have shown that the change of
the nature of the catalytic acid/base can have a significant
[
14,15]
effect on the reaction mechanism.
However, the effect of
active-site spacial changes by residue substitution needs to be
considered. For example the lack of ketoreduction of wild-
type IPR with 3a and 3b may be due to a preference for
binding in a conformation consistent with double bond
reduction, while the steric bulk of Tyr in IPR variant E238Y
may orient the substrate in a position suitable for keto-
reduction. Further studies will be needed to determine the
relative contribution of catalytic residue type vs. steric
constraints in determining the overall mechanism of the
catalysis.
5
a–d), however it performed minor ketoreduction with
substrate 3a to form the equivalent alcohol products 8a
Table 2, entries 1 and 2). Additionally it showed MNMR-like
activity towards Mentha compounds 1a,b, forming primarily
b and 2d, respectively (Table 2, entries 3–6), although the
(
2
product yields and enantiopurity were lower than with wild-
type MNMR. Interestingly, reactions with 1b at pH 7.0
generated a slightly higher yield of products, but they were
obtained in near racemic form (Table 2, entry 6. Therefore,
replacing of active-site Glu by Tyr has converted the enzyme
from an ene reductase into a ketoreductase, albeit with lower
catalytic efficiency and enantiospecificity.
We have pinpointed a simple mechanistic switch between
ene-reductase and ketoreduction activity in the SDR super-
family. This simple mechanistic switch, in addition to other
residue substitutions to improve catalytic efficiency, could
potentially transform SDR ketoreductases into novel ene
reductases and provide attractive routes to novel ene-
reductase catalysts. This would reduce the dependence on
traditional FMN-containing OYEs for the biocatalytic reduc-
tion of a,b-unsaturated alkenes and complications (reaction
rates, yields, and product enantiopurity) that arise when
In the case of MNMR variant Y244E, ketoreduction was
not seen with any substrate tested (1a,b, 3a,b, and 5a–d).
Minor double bond reduction was detected with substrate 5c
to form 6c (Table 2, entry 7). MMR and MNMR are known to
[
13]
OYEs are affected by molecular oxygen. Access to a new
class of ene reductases would open up the possibility of
developing new catalytic specificities typical of the SDR
superfamily for the reduction of a,b-unsaturated alkenes.
[
1a]
have narrower substrate specificities than IPR (Table 1 and
Figure S4), suggesting further mutations are required to form
a more active ene reductase.
Interestingly, studies with mechanistically different
enzymes of the class I aldolase family (transaldolase and
Acknowledgements
Table 2: Biocatalytic reduction of cyclic ketones by enzyme variants
IPR E238Y and MNMR Y244E.
[
a]
We thank Syed T. Ahmed (University of Manchester) for his
assistance in the synthesis and analysis of product standards,
Diamond Light Source for access to beamlines I02, I03, and
I04 (proposal numbers mx8997 and mx12788), and Dr. Colin
Levy, Manchester Protein Structure Facility (MPSF), for help
with X-ray data collection. This work was funded and
supported by the UK Biotechnology and Biological Sciences
Research Council (BBSRC BB/J015512/1 and BB/M000354/
1), the Centre for Synthetic Biology of Fine and Speciality
Chemicals (SynBioChem; BBSRC: BB/M017702/1), the
Centre of Excellence for Biocatalysis, Biotransformations
and Biocatalytic Manufacture (CoEBio3; University of Man-
chester), and GlaxoSmithKline. N.S.S. was a Royal Society
Wolfson Merit Award holder and is an Engineering and
Physical Sciences Research Council (EPSRC; EP/J020192/1)
Established Career Fellow.
[
b]
[b]
Entry
Enzyme
Substrate Product Yield [%] ee [%]
1
2
3
4
5
6
7
pH 6 IPR E238Y
pH 7 IPR E238Y
pH 6 IPR E238Y
pH 7 IPR E238Y
pH 6 IPR E238Y
pH 7 IPR E238Y
3a
3a
1a
1a
1b
1b
8a
8a
2b
2b
2d
2d
6c
<1
<1
38
42
33
47
3
nd
nd
[
[
[
[
c]
c]
d]
d]
45 (1S,2S,5R)
46 (1S,2S,5R)
47 (1R,2S,5S)
rac
pH 7 MNMR Y244E 5c
nd
[
a] Reactions (1 mL) were performed in buffer (50 mm KH PO pH 6.0
2 4
for IPR; 50 mm Tris pH 7.0 for MNMR and IPR) containing mono-
terpenoid (1a,b, 3a,b, and 5a–d; 5 mm), enzyme (5 mm or 10 mm for IPR
and MNMR, respectively), NADP (10 mm), glucose (15 mm), GDH
Keywords: biotransformations · isopiperitenone reductase ·
Mentha essential oil biosynthesis · short-chain dehydrogenases/
reductases · structure elucidation
+
(
10 U), and enzyme (2 mm). The reaction solutions were agitated at 258C
for 24 h at 130 rpm. Product identification was performed by both
comparing retention times with authentic standards and identification
by GCMS on a DB-WAX column (only GCMS identification for product
8
a). Figure S10 gives the GCMS spectra traces of the additional products
and their respective substrates. [b] Product yield and enantiomeric
excess were determined by GC analysis using DB-WAX and Chirasil-DEX-
CB columns, respectively. nd=not determined due to low product yield.
[c] Other isomer formed (20% yield) was 2a. [d] Other isomer formed
(
2% yield) was 2c.
4
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
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