Metagenomic ene-reductases for the bioreduction of sterically challenging enones

Article abstract of DOI:10.1039/c9ra06088j

Ene-reductases (ERs) of the Old Yellow Enzyme family catalyse asymmetric reduction of activated alkenes providing chiral products. They have become an important method in the synthetic chemists' toolbox offering a sustainable alternative to metal-catalysed asymmetric reduction. Development of new biocatalytic alkene reduction routes, however needs easy access to novel biocatalysts. A sequence-based functional metagenomic approach was used to identify novel ERs from a drain metagenome. From the ten putative ER enzymes initially identified, eight exhibited activities towards widely accepted mono-cyclic substrates with several of the ERs giving high reaction yields and stereoselectivities. Two highly performing enzymes that displayed excellent co-solvent tolerance were used for the stereoselective reduction of sterically challenging bicyclic enones where the reactions proceeded in high yields, which is unprecedented to date with wild-type ERs. On a preparative enzymatic scale, reductions of Hajos-Parish, Wieland-Miescher derivatives and a tricyclic ketone proceeded with good to excellent yields.

Full text of DOI:10.1039/c9ra06088j

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Metagenomic ene-reductases for the bioreduction
of sterically challenging enones†
Cite this: RSC Adv., 2019, 9, 36608
Dragana Dobrijevic,‡a Laure Benhamou, ‡b Abil E. Aliev,b Daniel Mendez-Sanchez,b
´
´
Natalie Dawson,c Damien Baud,b Nadine Tappertzhofen,b Thomas S. Moody,d
b
a
Christine A. Orengo,c Helen C. Hailes
and John M. Ward
*
*
Ene-reductases (ERs) of the Old Yellow Enzyme family catalyse asymmetric reduction of activated alkenes
providing chiral products. They have become an important method in the synthetic chemists' toolbox
offering a sustainable alternative to metal-catalysed asymmetric reduction. Development of new
biocatalytic alkene reduction routes, however needs easy access to novel biocatalysts. A sequence-
based functional metagenomic approach was used to identify novel ERs from a drain metagenome.
From the ten putative ER enzymes initially identified, eight exhibited activities towards widely accepted
mono-cyclic substrates with several of the ERs giving high reaction yields and stereoselectivities. Two
highly performing enzymes that displayed excellent co-solvent tolerance were used for the
stereoselective reduction of sterically challenging bicyclic enones where the reactions proceeded in high
yields, which is unprecedented to date with wild-type ERs. On a preparative enzymatic scale, reductions
of Hajos–Parish, Wieland–Miescher derivatives and a tricyclic ketone proceeded with good to excellent
yields.
Received 5th August 2019
Accepted 2nd November 2019
DOI: 10.1039/c9ra06088j
rsc.li/rsc-advances
of two chiral centers via a formal addition of hydrogen across
a double bond.4 On an industrial scale, processes typically
Introduction
involve a transition metal complexed to a chiral ligand. Even
though these methods achieve good selectivities and yields,
catalyst removal is oen challenging. Biocatalytic alkene
reductions with ene-reductases (ERs) offer an alternative with
potential industrial applications.5–7 ERs reduce alkenes acti-
vated with an electron-withdrawing group and use a co-factor
(Fig. 1A). Four different classes of ERs have been explored for
applications in biocatalysis, which differ in their stability,
reaction mechanism, substrate scope, diastereomer-, and/or
enantioselectivity. The most extensively investigated class of
ERs is the Old Yellow Enzyme (OYE) family of NAD(P)H
dependent avin containing oxidoreductases (EC 1.6.99.1)8–10
that catalyse the reduction of a,b-unsaturated compounds
activated with keto, aldehyde and nitro moieties.11 Although
OYEs have been employed on industrially relevant substrates,
such as in the synthesis of high value compounds including
pregabalin12 and menthol,13,14 wider industrial implementation
has proved challenging. ERs suffer a number of limitations such
as low tolerance to organic co-solvents, low substrate loading
tolerance and a narrow substrate range with few reports
describing the use of bulky substrates.5 Therefore, access to
novel, robust and highly active enzymes is vital to develop bio-
catalytic routes using ERs for larger scale applications.
Over the last 30 years, regulations from the FDA and the EMA
have become more stringent in relation to product specica-
tions requiring higher optical purities for chiral drug
substances.1 In addition, the substantial growth in demand for
chiral drug substances in new chemical entity pipelines has
urged synthetic chemists to discover more efficient and
sustainable asymmetric transformations to afford enantiopure
molecules.2 Biocatalysis is frequently a methodology of choice
to achieve selective transformations with minimal environ-
mental impact.3 Biocatalytic transformations are generally
performed in aqueous-based media with enzymes acting as
chiral catalysts. Enzymes exhibit excellent chemo-selectivities,
avoiding the need for protecting groups, and possess high
asymmetric induction potential. In synthetic methodology, the
asymmetric reduction of alkenes allows the possible formation
aDepartment of Biochemical Engineering, University College London, Bernard Katz
Building, London WC1H 6BT, UK. E-mail: j.ward@ucl.ac.uk
bDepartment of Chemistry, University College London, 20 Gordon Street, London,
WC1H 0AJ, UK. E-mail: h.c.hailes@ucl.ac.uk
cStructural and Molecular Biology, University College London, London, WC1E 6BT, UK
dAlmac, Department of Biocatalysis & Isotope Chemistry, 20 Seagoe Industrial Estate,
Craigavon, BT63 5QD, N. Ireland, UK
To date, novel ERs have been identied from bacteria, fungi
and plants mainly by conventional microbial screening,
genome mining and more recently by a protein data bank
† Electronic supplementary information (ESI) available: Procedures for the
enzyme discovery, biotransformations, molecular docking and analytical data of
all compounds. See DOI: 10.1039/c9ra06088j
‡ These authors contributed equally.
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database.22 To discover novel metagenomic ERs, predicted
protein sequences assigned to the Pfam entry PF00724 were
analyzed. According to Pfam, enzymes in this family are NADH
avin oxidoreductase/NADH oxidase that contain a TIM-barrel
fold and commonly use FMN/FAD as a cofactor.22 Further-
more, we found that all 63 published and biochemically char-
acterized prokaryotic ERs from the Old Yellow Enzyme family
belonged to this entry.9 In the drain metagenome, from a total
of 38 predicted proteins assigned to PF00724, 14 were encoded
by full-length genes and 24 by truncated genes. When
sequencing reads were assembled into contigs, some of the
predicted ORFs extended beyond the boundaries of assembled
contigs resulting in N- or C-terminally truncated genes.
Although PCR amplication of nearly full-length genes is
possible by designing the primer sets based on homologue
sequence regions available in public databases, it was decided
to focus on the analysis of the full-length genes and their cor-
responding proteins. Multiple sequence alignments of 14 full-
length putative ERs with selected characterized members of
the OYE protein family showed that active site residues8 were
conserved in 10 of the metagenomic proteins (Fig. S1†). These
ten putative ERs were taken forward as candidates for cloning
and functional characterization. The other four proteins were
identied as possible oxygen-sensitive enoate-reductases (EC
1.3.1.31) based on sequence analysis and were not investigated
further in this work.
A total of nine out of ten genes encoding putative ERs were
successfully amplied directly from the drain metagenomic
DNA, cloned with a C-terminal His6 tag and named with
plasmid numbers: pQR1439, pQR1440, pQR1442, pQR1443,
pQR1445, pQR1446, pQR1907, pQR1908, pQR1909. The nine
novel ERs shared sequence identity of between 76% and 100%
to protein sequences in the NCBI database (Table S1†). The
closest homologues of the metagenomic ERs belonged to
different bacterial species, with all coming from Proteobacteria
phylum, the predominant phylum in the drain metagenome.
Multiple sequence alignments of the nine ERs showed
a sequence identity ranging from 22% to 54%. Based on
differences in conserved sequence residues the majority of the
ERs were assigned to subclasses of OYEs previously described as
“classical” and “thermophilic-like” (Fig. S1†).8 ERs pQR1439,
pQR1443 and pQR1446, however showed differences in the
conservation of distinct residues. Phylogenetic analysis
(Fig. S1†) further indicated that ERs pQR1907, pQR1908,
pQR1445, and pQR1440 belonged to the “classical” group
together with OYEs from Saccharomyces pastorianus and
Saccharomyces cerevisiae, NCR from Zymomonas mobilis, and
pentaerythritol tetranitrate reductase (PETNR) from Enter-
Fig. 1 (A) Reduction of activated alkenes catalysed by ERs from the
OYE family, (B) overview of this study.
search guided by catalytic site structural information.5,15–17 Next-
generation sequencing technologies and omics approaches
have opened up the opportunity to speed up the discovery of
useful biocatalysts by providing access to unexplored environ-
mental microbial communities.18
In a previous study, we have reported a sequence-based
functional metagenomics approach to identify novel bio-
catalysts.19,20 Microbial communities from the human oral
cavity and more recently from a domestic drainpipe were
investigated.19–21 The drain proved to be an interesting niche
providing a highly valuable dataset for bioprospecting and led
to the identication of native enzymes with notable character-
istics, such as organic solvent stability oen only observed in
highly engineered enzymes.21 In the present study, in silico
searches of the drain metagenome led to the identication of
eight novel and active ERs from the OYE family (Fig. 1A) of
which two ERs displayed high activity with bi- and tri-cyclic
enones (Fig. 1B), on a preparative scale, to generate chiral syn-
thons in good to excellent yields and stereoselectivities.
Results and discussion
Drain metagenome and the identication of ERs
Metagenomic DNA was isolated from a sample collected from obacter cloacae, while pQR1909 and pQR1442 belonged to
a domestic shower drainpipe and sequenced by the Illumina ‘thermophilic-like’ ERs populated with mesophilic (YqjM from
MiSeq platform. De novo assembly of the drain metagenome Bacillus subtilis) as well as thermophilic (TOYE from Thermoa-
resulted in 69 962 contigs with sizes ranging from 500 base naerobacter pseudoethanolicus) enzymes.8,9 Three remaining ERs
pairs (bp) to 67 494 bp and a mean length of 1500 bp. Analysis clustered apart from these two OYE groups, expanding further
revealed that 106 954 open reading frames (ORFs) were pre- newly proposed subclasses in the OYE family phylogenetic
dicted with 39 915 genes being longer than 600 bp (see ESI†). tree.9,39 pQR1443 and pQR1446 clustered together with Chr-
Approximately 50% of the predicted ORFs had functional OYE1 from Chryseobacterium sp. while pQR1439 clustered
domains that could be annotated by the Pfam Protein Family close to Nox from Rhodococcus erythropolis and YqiG from
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Bacillus subtilis (Fig. S1†).39 All the metagenomic ERs were
heterologously expressed in E. coli BL21 (DE3). The ER NCR has
been well studied and successfully used with a range of linear
and monocyclic substrates and was therefore used as a refer-
ence enzyme.23
Table 2 Reduction of carvones using the ERs pQR1907, pQR1445 and
NCRa
Initial screening of metagenomic ERs
To screen the panel of nine metagenomic ERs, puried enzymes
were initially used (Fig. S2†). A qualitative spectrophotometric
assay with substrates 1 and 2 that are readily accepted by the
OYEs,23 indicated NADPH-dependent reductase activity for
seven of the enzymes. No or low activity was observed for the
enzymes expressed from pQR1439 and pQR1443 respectively
(Table S2†). To further assess the ERs, activity was quantied
using GC-analysis with substrates 1 and 2 to give 3 and 4
respectively, NADPH and glucose-6-phosphate dehydrogenase
(G6PDH) to recycle the co-factor in situ and glucose-6-phosphate
sodium salt (G6PNa) as co-substrate. Seven metagenomic ERs
gave high yields (by GC analysis) with both substrates (Table 1),
with higher activities towards compound 2 for all enzymes.
As observed in the spectrophotometric assay, the ER
expressed from pQR1443 showed low activity, while no activity
was observed with the ER from pQR1439; these two enzymes
were not used in further experiments. For the active ERs,
products were obtained in moderate to high stereoselectivities
(83–99% ee) for the R enantiomer (Table 1). With NCR, yields
and the selectivity obtained for the reduction of 1 and 2 were in
agreement with reported values.23
(2R,5S)-6
(2R,5R)-7
ER
Yield [%]
Yield [%]
pQR1907
pQR1445
NCR
82
81
78
66
n.d.
63
Substrate (10 mM), puried ER (0.7–0.8 mg mLꢁ1), NADP+ (2.8 mM),
G6PDH (20 U), G6PNa (100 mM), in Tris–HCl (50 mM) and DMSO
(10%) at pH 7.4, 30 ꢂC, 1 h, 300 rpm. Reactions were performed in
triplicate. Yields and ees were determined by GC analysis; n.d. not
determined.
a
Next, the industrially important substrate carvone 5 was
tested. Reduction products of 5, (2R,5S)- and (2R,5R)-dihy-
drocarvones give access to chiral building-blocks for the
synthesis of natural products (e.g. striatenic acid, pechueloic
acid), antimalarial drugs, fragrances and bio-renewable poly-
esters.24 The bioreduction of carvone with ERs has been reported,
with both carvone enantiomers converted in high yield and
selectivity.6,25,26 Initial assays using GC analysis and the previously
optimised reaction conditions with a reaction time of 20 h
resulted in low yields (ꢀ20–30%) of dihydrocarvones 6 and 7 for
selected ERs (Fig. S3†). No background reaction was observed in
the absence of enzyme. Reducing the reaction time to 3.5 h
increased the yields to ꢀ40–60% (Fig. S4†). For the reduction of
R-5, lower yields were probably due to previously reported solu-
bility issues.27 The formation of 6 was then optimised further
with the enzymes expressed by pQR1907 and pQR1445 as they
were readily expressed and gave rise to high yields in the reduc-
tion of substrates 1, 2 and 5. Interestingly, S-5 was reduced to 6 in
1 h (Fig. S5†) with ERs from pQR1907 and pQR1445 in 82% and
81% yields, respectively, with complete selectivity for the (2R,5S)-
isomer (Table 2). Under these reaction conditions turnover
frequencies (TOFs) were high; pQR1907 achieved a high TOF of
722 hꢁ1 while pQR1445 reached 590 hꢁ1 molecules per hour,
better than many reported ERs and close to the best reported TOF
for (S)-carvone (z1000 hꢁ1), by LacER.37,38 The bioreduction of S-
5 with NCR provided the same selectivity in 78% yield and with
a TOF of 645 hꢁ1 in accordance with the literature.38 With the ER
from pQR1907 and NCR, isomer R-5 was reduced in 66% and
63% respectively, giving exclusively (2R,5R)-7.
Table 1 Bioreduction of activated ketones 1 and 2a
3
4
ERs
Yield [%]
Yield [%]
ee [%]
pQR1907
pQR1908
pQR1909
pQR1439
pQR1440
pQR1442
pQR1443
pQR1445
pQR1446
NCR
83
76
80
0
70
72
29
84
82
65
>99
90
>99
0
87
86
39
>99
98
90 (R)
99 (R)
92 (R)
—
92 (R)
90 (R)
83 (R)
99 (R)
83 (R)
99 (R)
69
Substrate (10 mM), puried ER (0.2–0.9 mg mLꢁ1), NADP+ (2.8 mM),
G6PDH (20 U), G6PNa (100 mM), in Tris–HCl (50 mM) and DMSO
(10%) at pH 7.4, 30 ꢂC, 20 h, 300 rpm. Reactions were performed in
triplicate. Yields and ees were determined by GC analysis. 1 and 2
remained unchanged in control reactions with no enzyme present.
a
Solvent tolerance of the enzymes from pQR1907 and pQR1445
Due to the high selectivity and yields towards the substrates
used, metagenomic ERs from pQR1907 and pQR1445 were co-
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cofactor rather than co-expression of a GDH. Another study41
used cyclohexenone and NADH with methanol and DMSO as
solvents and did not take into account co-factor recycling: while
some decrease in yields was observed at 30% levels of co-
solvent, 40% data was not included and very low yields were
observed at 50% levels.
In addition to the ERs, a recently reported wild type trans-
aminase originating from the same drain metagenome, but
from a different bacterium, showed extraordinary organic
solvent stability maintaining activity in up to 50% of DMSO
demonstrating that valuable enzyme properties can be obtained
from
metagenomic
samples
obtained
from
niche
environments.21
Preparative scale reactions and expansion of the substrate
scope
The two most promising ERs expressed from pQR1907 and
pQR1445, were further investigated for the reduction of more
complex and sterically challenging substrates including the
bicyclic Wieland–Miescher ketone 8. Decalones derived from 8
are versatile chiral building blocks in the synthesis of natural
products such as terpenoids and steroids.28 Little has been re-
ported on their biocatalytic reduction presumably due to
problems with substrate acceptance. Indeed, the bioreduction
of 8 has only been described in a patent using an industrially
engineered enzyme generated by gene shuffling of selected
OYEs.29
Due to the absence of a chromophore in the reduced
product, bioreductions of 8 were initially monitored by
following the depletion of the starting material. The reduction
of rac-8 and S-8 occurred with the complete conversion of the
starting material with the two ERs from pQR1907 and pQR1445
used as puried enzymes (Table 3). The reactions required
Fig. 2 Effect of co-solvent on the reduction of S-5. Substrate (10 mM),
co-expressed ER/G6PDH (1 mg mLꢁ1), NADP+ (2.8 mM), G6PNa (100
mM), in Tris HCl (50 mM) and co-solvent (%) at pH ¼ 7.4, 30 ꢂC, 1 h,
300 rpm. Reactions were performed in triplicate. Yields and the dia-
stereoselective ratio (d.r.) of 6 were determined by GC-analysis.
Standard deviations were below 5%.
expressed with the cofactor-recycling enzyme G6PDH from
Saccharomyces cerevisiae in E. coli BL21 (DE3) and prepared as
claried cell lysates. These co-expressed lysates were reacted
with S-5, and 6 was obtained in 95% yield (by GC analysis) with
the enzyme from pQR1907 (d.r. ¼ 95 : 5) and 96% yield (by GC
analysis) with the enzyme from pQR1445 (d.r. ¼ 97 : 3)
(Fig. S7†).
Table 3 Bioreductions of rac-8 and S-8 using the enzymes from
pQR1907 and pQR1445a
Activities towards S-5 were then investigated in the presence
of different concentrations of the water-miscible co-solvents,
DMSO and methanol (10–50% v/v) (Fig. 2). Both enzymes were
tolerant of up to 30% DMSO, aer which a signicant drop in
yields was observed. Methanol was also well tolerated with only
a slight loss of activity at a 30% level. Additionally, the reaction
selectivity decreased from d.r. ¼ 95 : 5 to d.r. ¼ 88 : 12 for the
enzyme from pQR1907 and from d.r. ¼ 97 : 3 to d.r. ¼ 88 : 12 for
the enzyme from pQR1445 when using S-5 and higher amounts
of methanol, but little change in the selectivity was observed
when using up to 40% of DMSO.
This high-co-solvent tolerance of both the ERs and the
cofactor recycling enzyme is notable and highlights their
potential utility. The use of DMSO in up to 50% v/v has been
used with ERs40 but much lower conversions (and ees) were
observed at greater than 20% v/v DMSO. No methanol was used
in that study. In addition, that report added GDH to recycle the
S-8
Rac-8
ER
Conv. [%]
Conv. [%]
pQR1907
pQR1445
>99
>99
>99
97
Substrate (10 mM), puried ER (0.2–0.9 mg mLꢁ1), NADP+ (2.8 mM),
G6PDH (20 U), G6PNa (100 mM), in Tris–HCl (50 mM) and DMSO
(10%) at pH ¼ 7.4, 30 ꢂC, 20 h, 300 rpm. Reactions were performed in
triplicate. Conversions were determined by HPLC based on the
depletion of starting material.
a
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Table 4 Bioreductions on preparative scalea
Substrate
Enzyme (mg mLꢁ1
)
Product
Isolated yield (%)
95b
d.r.
pQR1445 (0.5)
98 : 2
pQR1907 (1)
pQR1907 (2)
pQR1907 (2)
pQR1907 (2)
95b
71b,c
90d
90d
Syn only
Syn only
58 : 42
85 : 15
pQR1907 (1)
pQR1907 (4)
94b
50d
Syn only
—
a
Substrate (5 mM for rac-16; 10 mM for S-5 and R-11 20 mM for S-8, S-11, rac-11, S-14), co-expressed ER/G6PDH (1–4 mg mLꢁ1), NADP+ (2.8 mM),
G6PNa (100 mM), in Tris–HCl (50 mM) and DMSO (10%) at pH ¼ 7.4, 30 ꢂC, 300 rpm. b Isolated yield. c Purication by column chromatography.
d
2.5 mL scale. Quantication by NMR using an internal standard (1,3,5-trimethoxybenzene).
longer times to proceed compared to the carvone substrates and selectivity for the syn-isomer at the ring junction. Compound 9
were run for 20 hours. A kinetic study with rac-8 revealed a faster was also obtained in high purity aer extraction from the
depletion of the S-enantiomer with puried pQR1907 (Fig. S6†). reaction mixture with ethyl acetate, without the need for any
From this promising data, ERs from pQR1907 and pQR1445, further purication. Under the same reaction conditions the
and NCR, for comparison purposes, co-expressed with the performance of NCR was inferior to these novel ERs affording 9
recycling system G6PDH, were used as claried cell lysates with in only 16% yield.
S-5 and S-8 and the reactions conditions optimized (Fig. S7 and
S8†). Biotransformations were then carried out on a preparative
25 mL scale with the initial concentration of 10 mM of S-5 and
20 mM of S-8 (see Fig. S8 and S9†). Under these conditions the
ER expressed from pQR1445 fully converted S-5 in an hour
affording (2R,5S)-dihydrocarvone 6 in 95% yield with only traces
of the other diastereomer (NMR: d.r. ¼ 98 : 2) (Table 4). The
product 6 was readily isolated by extraction from the reaction
mixture with ethyl acetate without further purication. The
biocatalytic preparative scale reaction with the enzyme
expressed from pQR1907 on S-8 resulted in a 95% yield of dione
Fig. 3 (A) Wieland–Miescher ketone S-8 docked in the active site of
the ER pQR1907 homology model (residues coloured beige) using
Autodock Vina.32 (B) Key residues surrounding the substrate S-8 (col-
9 in 20 hours. The selectivity and the stereochemistry were
determined by 1H NMR spectroscopy (J-values) (Fig. S10–S16†).
Analysis revealed that the reduction occurred with complete oured white) and FMN cofactor (coloured green). Images generated
using Chimera.33
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In order to understand better the enzyme performance, these types of substrates have not been reported with wild type
molecular modelling was performed using a homology model of enzymes. Sterically challenging substituted-WMKs 11 and
the ER pQR1907.30,31 Docking of the substrate S-8 into the active a tricyclic derivative 16 were also converted in good yield and
site was performed and its proximity to the FMN co-factor and selectivity adding novelty to the substrate scope known for ene-
the key-residues were determined (Fig. 3) (Table S3†). Notably, reductases. The metagenomic ERs have signicant potential in
S-8 readily tted into the active site close to the FMN co-factor synthetic applications and could also serve as an excellent
˚
(distance from the alkene to the nitrogen of the FMN ꢀ3.6 A) starting point for future protein engineering.
˚
providing the reductive capability. Also, the alkene was ꢀ3.7 A
from the key Tyr186 residue for protonation. In contrast,
docking of S-8 into the active site of NCR (crystal structure
Conflicts of interest
4A3U) did not result in a productive mode. These results could There are no conicts to declare.
help to rationalize the discrepancy of reactivity between the two
ERs with S-8, where it is readily accepted by the ER from
pQR1907, but only low levels of product were formed with NCR.
Acknowledgements
Informed by the docking results the substrate scope was Funding from the Biotechnology and Biosciences Research
further explored towards other bicyclic enones using the ER Council (BBSRC) and ALMAC for D. D., L. B., D. M.-S., D. B.,
expressed from pQR1907 (Table 4). Compound S-11 was (BB/L007444/1), (BB/N01877X/1) are gratefully acknowledged.
prepared using established methods (90% ee),34 and was effi- We also acknowledge equipment support from EPSRC
ciently reduced affording 12 in 71% yield as the syn isomer (EP/P020410/1).
(aer column chromatography). Rac-11 was also converted into
a mixture of 12 : 13 in 90% yield and the reduction afforded
a mixture of isomers (d.r. ¼ 58 : 42), highlighting that R-11 was
Notes and references
converted more slowly. This rate discrepancy could result from
preferred stereochemistries in the ER active site. However, by
decreasing the substrate loading to 10 mM, R-11 (84% ee) was
fully converted to the dione 13 with a high selectivity for the
compound with the trans ring junction (Fig. S15 and S16†). In
addition, the Hajos–Parrish ketone S-14 could also be readily
reduced to give 15 in 94% yield. The product was also isolated
by extraction from the reaction mixture with ethyl acetate and
1 W. H. Brooks, W. C. Guida and K. G. Daniel, Curr. Top. Med.
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2 V. Farina, J. T. Reeves, C. H. Senanayake and J. J. Song, Chem.
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3 R. A. Sheldon and J. M. Woodley, Chem. Rev., 2018, 118, 801–
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4 H. U. Blaser, in Applications of Transition Metal Catalysis in
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2012, pp. 315–342.
1
no purication step was required. The H NMR spectroscopic
data of 15 revealed total selectivity in the reaction for the syn
isomer. Finally, taking advantage of the metagenomic enzyme
to tolerate higher concentrations of DMSO, a bulkier tricyclic
enone rac-16 was also synthesized.35 Remarkably it was also
accepted to give the reduced product rac-17 in 50% yield. This
suggests that the presence of the aromatic ring may enable both
faces of the C]C bond in 16 to be presented to the FMN co-
factor. Such reductive reactivity with truncated steroids has
not been reported before with wild type ERs and is reminiscent
of the activity exhibited by androstenedione reductase, a 3-oxo-
5-alpha-steroid 4-dehydrogenase family protein, which shares
only 9% protein sequence identity with pQR1907.36
5 H. S. Toogood and N. S. Scrutton, ACS Catal., 2018, 8, 3532–
3549.
´
6 C. K. Winkler, G. Tasnadi, D. Clay, M. Hall and K. Faber, J.
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