Facile Stereoselective Reduction of Prochiral Ketones by using an F420-dependent Alcohol Dehydrogenase

Article abstract of DOI:10.1002/cbic.202000651

Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor-dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F₄₂₀-dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self-sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F₄₂₀ or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions.

Full text of DOI:10.1002/cbic.202000651

Combining Chemistry and Biology
Accepted Article
Title: Facile Stereoselective Reduction of Prochiral Ketones using an
F420-dependent alcohol dehydrogenase
Authors: Caterina Martin, Gwen Tjallinks, Milos Trajkovic, and Marco
Fraaije
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To be cited as: ChemBioChem 10.1002/cbic.202000651
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1/2020

ChemBioChem
10.1002/cbic.202000651
COMMUNICATION
Facile Stereoselective Reduction of Prochiral Ketones using an
420-dependent alcohol dehydrogenase
F
Caterina Martin, Gwen Tjallinks, Milos Trajkovic and Marco W. Fraaije*
Abstract: Effective procedures for the synthesis of optically pure
alcohols are highly valuable. A commonly employed method involves
the biocatalytic reduction of prochiral ketones. This is typically
achieved using nicotinamide cofactor-dependent reductases. In this
work we demonstrate that a rather unexplored class of enzymes can
also be used for this. We used an F420-dependent alcohol dehydro-
genase (ADF) from Methanoculleus thermophilicus which was found
to reduce various ketones into enantiopure alcohols. The respective
S-alcohols were obtained in excellent enantiopurity (>99% e.e.).
Figure 1. Structural formula of the F420 deazaflavin cofactor. Also other
Furthermore, we discovered that the deazaflavoenzyme can be used
as a self-sufficient system by merely using a sacrificial cosubstrate
deazaflavins are indicated: FO and FOP.
(isopropanol) and a catalytic amount of cofactor F420 or the unnatural
cofactor FOP to achieve full conversions. This study reveals that
deazaflavoenzymes complement the biocatalytic toolbox for enantio-
selective ketone reductions.
While flavin and nicotinamide cofactors are readily available
together with a huge number of enzymes utilizing these cofactors,
the biocatalytic exploration of F420-dependent enzymes is lagging
behind. This is partly due to the fact that the deazaflavin cofactor
was difficult to obtain. The deazaflavin cofactor can be isolated
from F420-producing microbes, such as Mycobacterium smeg-
The biocatalytic production of chiral alcohols is feasible by em-
ploying different classes of enzymes: oxidoreductases (EC 1),
hydrolases (EC 3), and lyases (EC 4).[1] Several methods allow
for the production of chiral alcohols and an effective method
involves the enantioselective reduction of prochiral ketones by
alcohol dehydrogenases (ADHs). ADHs have gained great inter-
est due to their enantioselectivity, allowing for the development of
many biotechnological applications.[2] The applicability of ADHs is
somewhat limited due to their cofactor dependence: most ADHs
rely on the nicotinamide cofactors NADH or NADPH for perfor-
ming reductions.[3] Often, cofactor regeneration is achieved by
using an enzyme-coupled process.[4] We explored another type of
ADH: an F420-dependent ADH. Such type of ADH had never been
explored for biocatalytic reductions of ketones. The deazaflavin
cofactor F420 was discovered in 1972, first in methanogens and
later also in actinobacteria and other bacteria.[5–7] The redox
active part of the cofactor resembles the isoalloxazine ring of the
canonical flavin cofactors (Figure 1). The differences are the lack
of the N5, different groups on the benzylic moiety, and the poly-γ-
glutamate lactyl tail instead of the adenine part of FAD. The
absence of N5 (a carbon instead) dictates that the cofactor is an
obligatory hydride transfer agent, similar to the nicotinamide
cofactors. The F420 cofactor displays an absorption maximum at
420-producing recombinant microorganism.[9,10]
matis or
a F
Although it involves a laborious process and yields small amounts,
it is nowadays available for biocatalytic studies. Furthermore, we
have recently demonstrated that a truncated version of the
cofactor, lacking the poly-γ-glutamate lactyl tail (FOP, see Figure
), is also accepted as cofactor by F420-dependent enzymes.[11]
1
FOP represents an unnatural deazaflavin cofactor for which a
relatively easy synthetic procedure has been developed. Another
bottleneck for using F420-dependent enzymes has been the lack
of knowledge of and access to such biocatalysts. Particularly,
these redox enzymes seem promising for performing selective
reductions by virtue of the unique low redox potential deazaflavin
cofactor. This would require regeneration of the reduced deaza-
flavin cofactor. This demand for F420H -regenerating enzymes has
2
been satisfied by the recent discovery of several deazaflavo-
enzymes. The availability of several F420-dependent glucose-6-
phosphate dehydrogenases is especially attractive for this.[12]
In the last decade the interest in exploring deazaflavoenzymes for
biocatalysis has increased.[8,13–15]
F420-dependent enzymes were
shown to be involved in the production of antibiotics, and could
be used for the asymmetric reduction of prochiral imines and
enones.[16–20] In this work we explored an F420-dependent ADH
4
20 nm, hence its name. The redox potential of this redox cofactor
(
ADF) for its ability to perform enantioselective reductions of pro-
is exceptionally low: –380 mV [(220 mV for FAD and –320 mV
NAD(P)].[5,8]
chiral ketones. The F420-dependent alcohol dehydrogenase from
Methanoculleus thermophilicus was chosen for this. ADF is a
relatively small enzyme (37 kDa) which was first described in
1
989. It can be recombinantly produced in Escherichia coli and its
C. Martin, G. Tjallinks, Dr. M. Trajkovic, Prof. Dr. M. W. Fraaije
Molecular Enzymology Group, University of Groningen
Nijenborgh 4, Groningen (The Netherlands)
E-mail: m.w.fraaije@rug.nl
[17,21]
crystal structure has been elucidated.
Previously it was
shown that ADF converts small aliphatic secondary alcohols into
ketones. It was shown that during this oxidation step a hydride is
transferred in a stereoselective manner from the alcohol to the Si-
face of the F420 cofactor (to the C5 atom).[ It was demonstrated
that the enzyme is able to oxidize isopropanol to acetone and the
Homepage: https://fraaije.info/
22]
Supporting information for this article is given via a link at the end of
the document.
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ChemBioChem
10.1002/cbic.202000651
COMMUNICATION
reverse reaction.[17] This was a clear indication that the enzyme
may act as an enantioselective ketone reductase through an
enantioselective hydride transfer.[21]
alcohols. For most ketones, excellent enantioselectivity was
observed (>99% e.e.), except for substrate 3 (92% e.e.) and
substrate 6 (racemic product) (Table 1).
First, we explored the substrate scope of ADF to find out whether
it is able to accept alcohols different from isopropanol. Alcohol
oxidation activity could be easily monitored by measuring the
decrease of absorbance of F420 (the reduced form does not absorb
at 420 nm). ADF did not convert the following alcohols: 2-heptanol,
Table 1. Reduction of prochiral ketones by ADF [a]
.
Cofactor
Conversion
(%)
Entry Substrate Cofactor
e.e. (%)
regeneration [
b]
5
-amino-1-pentanol, 1-amino-2-propanol, 3-chloro-1,2-propane-
1
EF420
1
1
F
420
420
FGD
-
96
95
>99 (S)
>99 (S)
diol, 1-phenyl-1,2-ethanediol, 1-indanol, acetoin, carveol, cyclo-
hexylmethanol, tetrahydropyran-2-methanol, 3-butyn-2-ol. Never-
theless, we could identify a set of aromatic and aliphatic alcohols
that were not reported before as substrate of ADF. The corres-
ponding ketones were investigated as prochiral substrates for
ADF (Figure 2).
1IF420
F
1
IFop
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
FOP
-
5
n.d.*
>99 (S)
>99 (S)
n.d.*
2EF420
F
420
420
FGD
77
2
IF420
IFop
EF420
F
-
80
2
FOP
-
3
3
F
420
420
FGD
>99
>99
94
92 (S)
72 (S)
>99 (S)
>99 (S)
>99 (S)
n.d.*
3
IF420
IFop
EF420
F
-
3
FOP
-
4
F
420
420
FGD
87
4
IF420
IFop
EF420
F
-
93
Figure 2. Ketones used for the biocatalytic exploration of ADF.
4
FOP
-
2
5
F
420
420
FGD
>99
>99
>99
>99
>99
>99
>99 (S)
>99 (S)
>99 (S)
0
5
IF420
IFop
EF420
F
-
5
FOP
-
6
F
420
420
FGD
6
IF420
F
-
-
0
6
IFop
FOP
0
[
a] Values obtained using 250 mM sodium phosphate pH 7.0, 10%
glycerol, 1% DMSO, 2.0 mM substrate, 2.0 mM ethyl benzene (internal
standard), 20 µM ADF, 40 µM F420 or 40 µM FOP. [b] For cofactor
regeneration, 20 mM glucose-6-phosphate with 10 µM FGD (‘FGD’) or
200 mM isopropanol (‘-‘) was used. Details are in the Supporting
Information. *Conversions were too low for an accurate determination
of e.e.
Figure 3. Coupled reactions for the reduction of prochiral ketones by ADF. ADF:
F
420-dependent alcohol dehydrogenase, FGD: F420-dependent glucose-6-phos-
phate dehydrogenase. (a) D-glucose-6-phosphate is used by FGD to regene-
rate F420 . (b) Self-sufficient use of ADF for ketone reductions with isopropanol
as cosubstrate to regenerate F420
H
2
In silico docking studies were performed to understand the mole-
cular basis for the (S)-stereoselectivity of ADF. For acetophenone
H
2
.
(
1) and methoxyacetone (5) docking suggested that the struc-
turally favorable product is in both cases indeed the S-enantiomer.
This can be explained by examining the role and position of the
active-site residues. Previous studies postulated that His39,
Trp43, Glu12 and Glu108 are crucial for substrate binding and
catalysis.[21] In the reduction reaction, a hydride transfer from the
For the first test conversions, we used the F420-dependent
glucose-6-phosphate dehydrogenase (FGD) from Rhodoccocus
jostii RHA1 (FGD) together with ADF (Figure 3a).[12] Gratifyingly,
it was found that the enzyme was able to fully and stereo-
selectively reduce methyl ketones to the corresponding (S)-
2
C5 atom of F420H to the ketone carbon with a simultaneous or
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ChemBioChem
10.1002/cbic.202000651
COMMUNICATION
stepwise proton addition from His39 to the ketone oxygen occurs.
Hence, the specific orientation of substrate towards the F420
cofactor and His39 is decisive in the enantioselective outcome of
the reaction. Docking of acetophenone revealed that Trp43
together with Val193, Trp229, Trp246, Cys249 and Phe255 form
a hydrophobic pocket which snugly accomodates the aromatic
ring of the substrate (Figure 4). As a result, acetophenone binds
in the active site in such a way that only the (S)-enantiomer can
be formed: the hydride can only be transferred to the Re-face of
the substrate. Positioning of acetophenone necessary to form the
was no difference between the use of glucose-6-phosphate
dehydrogenase and glucose-6-phosphate or merely isopropanol
for cofactor regeneration (Table 1). This shows that the use of
isopropanol as coupled substrate is a valid and simple alternative
to use ADF as enantioselective ketone reductase.
Finally, we also explored whether F420 can be replaced by an
unnatural deazaflavin cofactor: FOP. We have recently shown
that FOP can be prepared in a relatively easy manner and is often
accepted by F420-dependent enzymes.[11] The results obtained
using FOP as alternative cofactor with ADF and isopropanol as
cosubstrate revealed that ADF can also operate with this alter-
native deazaflavin (Table 1). Yet, the use of FOP resulted in lower
conversions for some of the ketones tested, while the enantio-
selectivity was largely retained. The inferior performance, when
compared with F420, probably reflects a poor recognition of FOP
by ADF.
(
R)-enantiomeric alcohol is prevented because its aromatic ring
cannot be accommodated in the active site in any other confor-
mation. Docking of methoxyacetone resulted in an analogous
optimal binding pose in which the apolar pocket next to the deaza-
flavin cofactor plays a crucial role in positioning the substrate such
that hydride attack will occur on the Re-face of the substrate,
assisted by proton transfer by His39. This will, again, only allow
formation of (S)-1-methoxy-2-propanol, as experimentally
observed.
In conclusion, the finding that ADF can reduce various prochiral
ketones in a highly (S)-stereoselective manner unveils a new
biocatalytically relevant class of enzymes: F420-dependent ketone
reductases. They can be regarded as alternatives to nicotinamide
cofactor-dependent enzymes.[23] Moreover, we demonstrate that
isopropanol can be used as cheap cosubstrate for cofactor
recycling, rendering ADF self-sufficient. With the crystal structure
of ADF available and many genes encoding for ADF homologs in
the genome sequence database, it will be exciting to explore other
variants for more demanding selective reductions.
Trp43
Cys249
Val193
W
Phe255
His39
Trp229
Trp246
Experimental Section
Figure 4 Binding pose of acetophenone (1) in ADF (PDB:1RHC). Left: overall
structure of ADF with docked substrate (cyan) and F420 (orange) highlighted in
sticks. Right: close-up of binding of acetophenone.
Reagents and chemicals were purchased from Sigma-Aldrich (St. Louis,
MO, USA) unless indicated otherwise. F420 was isolated from Mycobac-
terium smegmatis as described before.[9] The production strain M.
2
smegmatis mc 4517 was a kind gift from Dr. G. Bashiri from the University
The second aim of this work was to establish an efficient cofactor
regeneration system. While the used glucose-6-phosphate
dehydrogenase is effective in recycling F420H , such cofactor
2
of Auckland, New Zealand. Expression and purification of F420 dependent
enzymes are described in the Supporting Information.
For ketone reductions, reaction mixtures contained 200 µL of 250 mM
sodium phosphate pH 7.0, 10% glycerol, 1% DMSO, 2.0 mM substrate,
regeneration system still requires an addition enzyme and a
relatively expensive cosubstrate. Therefore, we explored whether
ADF can be used as reductase and dehydrogenase in one pot,
using a sacrificial and cheap alcohol as cosubstrate. This would
eliminate the need for another enzyme for cofactor recycling. For
this, we tested the use of isopropanol as it had been reported to
be a good ADF substrate. In order to determine the highest
concentration of isopropanol tolerated in conversions, we first
assessed the thermostability of ADF in the presence of different
amounts of isopropanol and its kinetic parameters with
isopropanol. ADF was found to be relatively tolerant towards
isopropanol with only a slight change in apparent melting
2.0 mM ethyl benzene (internal standard), 20 µM ADF, 40 µM F420 or 40
µM FOP, and 200 mM isopropanol or 20 mM glucose-6-phosphate with 10
µM FGD. The reaction was performed in a 1.5 mL Eppendorf tube in an
Eppendorf Thermomixer at 25 °C and 500 rpm for 24 h. The reaction was
extracted twice using 200 µL of ethyl acetate. The reactions passed over
anhydrous magnesium sulfate and finally analyzed using GC (details in the
Supporting Information).
Acknowledgements
temperature up to 200 mM isopropanol (T
6.0 °C, see Supporting Information). The steady-state kinetic
analysis confirmed that isopropanol is an effective substrate with
a K
value of 1.3 mM and a kcat of 1.7 s-1 (see Supporting
Information). A concentration of 200 mM of isopropanol was
selected to probe it as cosubstrate for the ADF-catalyzed
reduction of prochiral ketones. Remarkably, the use of
isopropanol as cosubstrate worked extremely well. In fact, there
m
went from 57.5 to
M.W.F and C.M received funding from the Dutch research council
NWO (VICI grant).
5
M
Keywords: biocatalysis • deazaflavin • enantioselective •
prochiral ketones • reduction
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Entry for the Table of Contents
An F420-dependent alcohol dehydrogenase can be used to perform asymmetrical reduction of prochiral
ketones. Conveniently, using a cheap sacrificial cosubstrate (isopropanol), regeneration of the
deazaflavin cofactor by the same enzyme can be achieved.
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