Exploiting the Catalytic Diversity of Short-Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope

Article abstract of DOI:10.1002/cbic.201800291

Numerous short-chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH-dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine-reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine-reducing enzymes.

Full text of DOI:10.1002/cbic.201800291

Accepted Article
Title: Exploiting Catalytic Diversity of Short-Chain Dehydrogenases/
Reductases: Versatile Enzymes from Plants with Extended Imine
Substrate Scope
Authors: Michael Müller, Sebastian Roth, Matthew Kilgore, and Toni
Kutchan
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To be cited as: ChemBioChem 10.1002/cbic.201800291
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10.1002/cbic.201800291
ChemBioChem
COMMUNICATION
Exploiting Catalytic Diversity of Short-Chain
Dehydrogenases/Reductases: Versatile Enzymes from Plants
with Extended Imine Substrate Scope
Sebastian Roth,[a] Matthew B. Kilgore,[b] Toni M. Kutchan,[b] and Michael Müller*[a]
Abstract: Numerous short-chain dehydrogenases/reductases
(SDRs) have found biocatalytic applications in C=O and C=C (enone)
reduction. For NADPH-dependent C=N reduction, imine reductases
(IREDs) have primarily been investigated for extension of the
substrate range. Here, we show that SDRs are also suitable for a
broad range of imine reductions. The SDR noroxomaritidine
reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis,
serving as an enone reductase. We have characterized NR using a
set of typical imine substrates and have established that the enzyme
is active with all four tested imine compounds (up to 99% conversion,
up to 92% ee). Remarkably, NR reduced two keto compounds as well,
highlighting this enzyme family’s versatility. Using NR as a template,
we have identified an as yet unexplored SDR from the Amaryllidaceae
Zephyranthes treatiae with imine-reducing activity (up to 95% ee). Our
results encourage the future characterization of SDR family members
as a means of discovering new imine-reducing enzymes.
oxidoreductases from different enzyme families can be easily
screened in search of an appropriate catalyst for a desired
transformation. Oxidoreductases that catalyze asymmetric
reductions are (sub)classified based on sequence similarity,
substrate scope, or cofactor dependency. Members of different
enzyme (sub)families can be suited for the same purpose.[9]
Most members of the short-chain dehydrogenase/reductase
(SDR) family belong to the EC class 1 (oxidoreductases). Despite
low sequence similarity between distinct members, these
enzymes display
a common scaffold that comprises the
Rossmann fold for binding of the cofactor NAD(P)H and facilitates
the above-noted proton and hydride transfer.[10] Thus, such
enzymes are putatively predestined for (imine) reduction.[11]
In a previous paper, we reported the promiscuous iminium
reductase activity of glucose dehydrogenase (GDH).[12] As this
enzyme is a prototypical SDR, we searched for further members
of this enzyme family that might be suited for imine reduction.
Some of us have recently identified noroxomaritidine reductase
(NR), an SDR from the plant Narcissus pseudonarcissus that is
involved in the biosynthesis of Amaryllidaceae alkaloids. NADPH-
dependent NR selectively reduces the C=C bond of the enone
moiety of noroxomaritidine (1). In addition, this SDR exhibits an
imine reductase activity with norcraugsodine (2), a precursor of 1,
albeit with a 1/400 of its native activity (Scheme 1).[13]
The biocatalytic reduction of C=N bonds is of rising importance as
an approach to chiral amines.[1] This transformation has almost
exclusively been the domain of imine reductases (IREDs), a class
of NADPH-dependent oxidoreductases, whose members
possess an extended substrate scope.[2] There are only a few
reports of other enzymes catalyzing imine reduction; nevertheless,
these enzymes usually display strict substrate specificity or their
substrate scope has not been investigated in detail.[1] From a
mechanistic point of view, one explanation for the particular
position of IREDs could be the lower intrinsic reactivity of C=N
bonds relative to C=O bonds towards nucleophiles. While IREDs
may have evolved a mechanism to overcome this obstacle,
details of their catalytic mechanism remain elusive, despite many
structural and kinetic studies.[2] Moreover, the ability of IREDs to
reduce imine substrates might be a promiscuous activity, as their
physiological function is not known.[3] Given the basic mechanism
of biocatalytic NAD(P)H-dependent reduction is the transfer of a
proton and a hydride regardless of the receiving electrophile,[4] we
expect that oxidoreductases other than IREDs would also be
applicable for imine reduction.[5] This idea is supported by the
recent work of Lenz et al. who optimized -hydroxy acid
dehydrogenases for imine reduction.[6]
Scheme 1. Involvement of noroxomaritidine reductase (NR) in Amaryllidaceae
alkaloid biosynthesis (modified from Kilgore et al.[13]).
Examples of enzymatic reduction promiscuity have been reported
for highly reactive iminium[12] and activated keto substrates.[14] NR,
however, displays catalytic versatility with non-activated
substrates, occurring in vivo.[13] This makes the enzyme an
interesting candidate to investigate its intrinsic versatility for the
biocatalytic reduction of imines. Here, we outline plant SDRs that
can reduce a broad range of imines, thereby expanding the
spectrum of imine-reducing enzymes. In addition, these enzymes
accept keto substrates and can serve as enone reductases. This
demonstrates that one and the same enzyme can possess the
ability to catalyze C=N, C=O, and C=C reduction.
For the asymmetric hydrogenation of C=O and C=C bonds,
biocatalysis is a beneficial and established tool.[7,8] A pool of
[a]
[b]
S. Roth, Prof. Dr. M. Müller
Institut für Pharmazeutische Wissenschaften
Albert-Ludwigs-Universität Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
E-mail: michael.mueller@pharmazie.uni-freiburg.de
Dr. M. B. Kilgore, Prof. Dr. T. M. Kutchan
Donald Danforth Plant Science Center
975 N. Warson Rd., St. Louis, MO 63132 (USA)
Supporting information for this article is given via a link at the end of
the document.
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The gene encoding NR was cloned in pET28a with an N-
terminal His tag. After overexpression in Escherichia coli BL21-
CodonPlus (DE3)-RP x pL1SL2, the enzyme was purified by Ni-
NTA affinity chromatography.[15] To obtain an overview of the
substrate range and stereoselectivity of NR-catalyzed reductions,
we chose previously described, representative imine compounds:
the well-known monocyclic 2-methyl-1-pyrroline (3), an
isoquinolinium compound (4), an exocyclic imine (5), and a
[a] Reaction conditions: 1 mg mL–1 SDR, 10 mM substrate, 20 mM D-glucose,
0.5 mM NADP+, 5 mM MgCl2, 0.25 mg mL–1 GDH, 30 °C, 20 h. Reactions with
4 and 8 were performed with NADPH (1 equiv) or contained malate dehydro-
genase/L-malate as cofactor regeneration system. [b] Conversions and de
values of 8a were determined by 1H NMR spectroscopy. [c] Conversions were
determined by HPLC analysis. [d] ee values were determined by chiral-phase
GC analysis. [e] ee values were determined by chiral-phase HPLC analysis.
[f] GC-MS analysis. – = not determined due to low conversion; n.d. = no
product detected; cis/trans = no significant excess of one diastereomer.
-carboline (6).[16–19] The two keto substrates
7 and 8
In the cases of more than 15% conversion, the enantiomeric
excess (ee) or diastereomeric excess (de) of the enzymatic
products 3a–8a was determined by chiral-phase GC or HPLC
analysis or 1H NMR spectroscopy (Table 1). NR reduced imine 4
with good and 6 with moderate ee values, and displayed different
stereoselectivity depending on the substrate. Moreover, NR
exhibited substantial conversion of ketones 7 and 8, although with
a low to moderate stereocontrol (Table 1).
complemented the substrate panel (Figure 1).
Encouraged by these results, we explored the catalytic scope
of NR homologues. Using the Basic Local Alignment Search Tool
(BLAST) with NR as a template, proteins with overall sequence
identity of 60–70% were found. The majority of the proteins
identified are annotated as tropinone-reductase-like SDRs. Such
enzymes typically accept keto compounds as substrates.[20]
Although a crystal structure of NR is available,[13] the prediction of
crucial amino acid residues for imine reduction remains
speculative. Therefore, we decided to take a biosynthesis-guided
approach and initially restricted the search to alkaloid-producing
organisms. It is known that members of the genera Pinus and
Picea contain alkaloids that might result from imine reduction.[21,22]
As we have shown that NR is capable of catalyzing such reactions,
we hypothesized that an SDR might be responsible for the
Figure 1. Structures of imines (3–6) and ketones (7, 8) tested as substrates in
this study.
With 5 mol% NADP+, a D-glucose/GDH system was applied for
cofactor regeneration. As GDH is active with isoquinolinium
compounds and also proved to be active with substrate 8,
reactions containing 4 and 8 were performed with NADPH in an
equimolar amount or with malate dehydrogenase/L-malate
cofactor regeneration. GC-MS analysis or 1H NMR spectroscopy
was used to analyze product formation, which indicated NR was
active with the tested imine and iminium compounds 3–6. The
assumed, as yet unknown, biosynthetic step(s). In
a
transcriptome of Pinus banksiana,[23] we located a candidate
(Pb_SDR, see Supporting Information) with 59.4% sequence
identity to NR. A BLAST search using this sequence as a template
revealed highly similar sequences from other Pinus/Picea species
for which alkaloids have been reported, followed by the above-
noted hypothetical tropinone-reductase-like SDRs. Hence, we
chose Pb_SDR as a candidate to be tested for imine reduction. In
addition, acetophenone (7) and (R)-3-methylcyclohexanone (8)
were tested. Ketone 8 has been identified as a substrate of
tropinone-reductase-like SDRs.[20] Based on the protein
sequence of Pb_SDR, a synthetic gene fragment was ordered,
which was codon-optimized for E. coli, and cloned into pET28a
via In-Fusion® cloning. After overexpression in E. coli BL21-
Gold(DE3), the N-terminally His-tagged enzyme was purified by
Ni-NTA affinity chromatography. Pb_SDR was active with
ketones 7 and 8 (Table 1), which were quantitatively converted.
The products were obtained with excellent ee [>99% (R)-7a] and
de (>99% trans-8a). Subsequently, we applied Pb_SDR for imine
reduction; however, no amine product was detected for
substrates 3–6. As an inherent inactivity of Pb_SDR was ruled out,
we examined the Pb_SDR sequence. For this purpose, we
combined information derived from the NR structure and from
literature concerning tropinone reductases[24] and applied it to
Pb_SDR, assuming a highly similar SDR fold. In tropinone reduct-
ase II from Datura stramonium, a histidine residue contributes to
charge distribution in the active site. Featured at the equivalent
position in Pb_SDR is an arginine residue (R107) whose perman-
ent positive charge might hinder the entrance/orientation of
1
conversion, determined by HPLC analysis or H NMR spectros-
copy, however, differed significantly (Table 1). While the mono-
cyclic (3) and exocyclic (5) compounds were poorly converted, our
data suggest a preference for polycyclic compounds 4 and 6,
which are structurally more similar to the physiological substrate
1; conversion of 4 (alongside malate dehydrogenase/L-malate)
and 6 surpassed 90%.
Table 1. Conversion of substrates 3–8 with SDRs and stereochemistry of the
resulting reduced products 3a–8a (in parentheses).[a]
Conversion [%], (enantiomeric or diastereomeric excess [%]
and absolute or relative configuration, respectively)
Enzyme
NR
3[b]
4[b,d]
5[c,e]
6[b,e]
7[c,e]
8[b]
10,
>99,
11,
90,
17,
>99,
–
(92, R)
–
(60, S)
(81, S)
(cis/trans)
Pb_SDR
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>99,
(>99, R) trans)
>99, (>99,
Pb_SDR_
R107N
traces[f]
n.d.
n.d.
>99,
(>99, R) trans)
>99, (~45,
Ao_SDR
Zt_SDR
traces[f]
>99,
(81, S)
>95, (~60,
cis)
14,
>99,
18,
87,
3,
46,
–
(60, R)
(95, S)
(50, S)
–
(cis/trans)
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(protonated) imine compounds in the enzyme’s active site. As
hypothesized, the rationally designed variant Pb_SDR_R107N
showed minimal, but measurable (GC-MS, Figure S6),
conversion of 5, while the ketoreductase capability remained
unchanged. This reveals that SDRs bear, in principle, the
prerequisites for ketone and imine reduction.
accept “common” ketones as substrates. This is remarkable as,
to date, promiscuous reductase activity of SDRs with C=N bonds,
and vice versa of IREDs with C=O bonds, has only been observed
with highly reactive compounds.[12,14] Along with the physiological
enone reductase activity of NR, this highlights the pluripotency
featured by some SDRs.[31] With regard to the tested substrates,
the reductase capability of Pb_SDR and Ao_SDR shifts towards
the keto compounds. Nevertheless, the four enzymes share the
SDR-typical catalytic triad and should have highly similar overall
structures.[10] Hence, the challenging question arises as to which
factors lead to the discrimination or the acceptance of keto and
imine compounds.
For the choice of the next candidate, we applied the following
criteria to scan the BLAST hits: sequences without a glycine
residue at the position equivalent to G192 in NR were excluded,
as this glycine is conserved in active SDR/tropinone
reductases.[20,25] Sequences with a basic residue at the equivalent
position to R107 of Pb_SDR were also excluded, as the positive
charge seems to hinder imine reductase activity. Moreover, the
candidate should feature at least one aromatic residue at the
equivalent positions to residues Y114 and F216 in the active site
of NR. These residues are presumably involved in substrate
binding and orientation, which might be reflected in the preference
of NR for the polycyclic substrates 4 and 6.[13] We identified a
sequence from Asparagus officinalis (Ao_SDR, see Supporting
Information) annotated as a tropinone-reductase-like SDR with a
sequence identity of 66% to NR and of 61% to Pb_SDR. Ao_SDR
was obtained as a His-tagged protein, analogously to Pb_SDR.
Ao_SDR was active with ketones 7 and 8 and, in contrast to wild-
type Pb_SDR, showed minimal conversion of compound 6 (Table
1, Figure S9). Hence, despite a relatively elevated sequence
similarity of Pb_SDR and Ao_SDR to NR, the activity profiles of
the wild-type enzymes differ. Whereas NR accepts both keto and
imine compounds, Pb_SDR and Ao_SDR seem to be almost
exclusively ketoreductases.
Experimental Section
The biotransformations were performed on an analytical scale (0.5 mL,
unless indicated otherwise) and the SDRs were applied as purified
enzymes. The substrates 3–8 were added from a stock solution (1 M in
MeOH), resulting in a 10 mM substrate concentration. Each reaction was
started by addition of a mixture (125 L) containing the other reaction
components dissolved in HEPES buffer (pH 7.5, 100 mM). The reaction
mixture consisted of purified SDR (1 mg mL–1), substrate (10 mM), NADP+
(0.5 mM), and the cofactor regeneration system: a) GDH (0.25 mg mL–1),
D-glucose (20 mM), and MgCl2 (5 mM) or b) malate dehydrogenase
(evocatal, 7.5 U), L-malate (50 mM), and MnCl2 (10 M). After incubation
for 20 h at 30 °C and 400 rpm, the reaction was stopped by removing the
enzyme (see the Supporting Information). For the analyses of product
formation and the determinations of enantiomeric/diastereomeric
excesses, see the Supporting Information.
Recently, the transcriptomic data from the 1000 Plants project
(1KP) were made publically available,[26–29] thus expanding the
data pool for finding new sequences to be tested. To identify
putative homologues of NR, the data of the included
Amaryllidaceae species were used for a BLAST search that gave
access to hits with a sequence identity of up to 88% to NR.
Applying the above-noted criteria, a hypothetical SDR from
Zephyranthes treatiae (Zt_SDR, see Supporting Information) was
chosen which displayed 87% overall sequence identity to NR.
Relative to NR, Zt_SDR showed slightly higher conversions of the
imine substrates 3 and 5 but was less active with the keto
Acknowledgements
We thank Lydia Walter and Marcel Wilde for helpful discussions,
Dr. Tobias Huber for providing substrate 6, Sascha Ferlaino for
measurement of 1H NMR spectra, and Dr. Kay Greenfield for help
in improving the manuscript. We acknowledge Dr. Dennis Wetzl
and Dr. Hans Iding from F. Hoffmann-La Roche, Ltd. for providing
substrate 4.
compounds
7 and 8 (Table 1). This demonstrates that
Keywords: alkaloids • asymmetric reduction • biocatalysis •
biosynthesis • imine reduction
homologues of NR can possess a comparable activity profile.
Prerequisite for this finding was the custom BLAST search on
transcriptomic data. Our results indicate that this approach is
appropriate for the identification of novel imine-reducing enzymes.
In addition, our finding is in line with the recent discovery of an
iminium-reducing SDR by plant genome mining.[30]
In summary, we have shown that members of the SDR
enzyme family are suitable to be applied as biocatalysts for imine
reduction, in addition to their well-established ability to reduce
C=O and C=C (enone) bonds. The SDR NR from N. pseudo-
narcissus is capable of reducing C=N bonds of imine/iminium
compounds with different scaffolds. With NR as a template and
using transcriptomic data for the BLAST search, we identified the
imine-reducing SDR Zt_SDR from the Amaryllidaceae Z. treatiae.
Thus, we have broadened the spectrum of imine-reducing
enzymes, underpinning our serendipitous findings for GDH.[12]
With the SDR family being one of the largest enzyme families,
these results suggest that it harbors additional enzymes with an
extended imine substrate scope. Moreover, NR and Zt_SDR also
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COMMUNICATION
One enzyme, three activities: The
plant SDR noroxomaritidine reductase
(NR) serves as an enone reductase in
alkaloid biosynthesis. Here, we show
that NR accepts different imine
compounds and features a keto
reductase activity. With NR as a
template, we have identified a novel
imine-reducing SDR.
S. Roth, M. B. Kilgore, T. M. Kutchan, M.
Müller
Page No. – Page No.
Exploiting Catalytic Diversity of
Short-Chain
Dehydrogenases/Reductases:
Versatile Enzymes from Plants with
Extended Imine Substrate Scope
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