Asymmetric reductive amination by a wild-type amine dehydrogenase from the thermophilic bacteria: Petrotoga mobilis

Article abstract of DOI:10.1039/c6cy01625a

The biocatalytic reductive amination of ketone to chiral amine is one of the most challenging reactions. Using a genome-mining approach, we found proteins catalyzing the reductive amination of ketones without a carboxylic function in the α or β position. The synthesis of (4S)-4-aminopentanoic acid (ee ≥99.5%) was achieved with the thermoactive amine dehydrogenase (AmDH) AmDH4 from Petrotoga mobilis in 88% yield. The high stability and substrate tolerance make AmDH4 a very good starting point for further discovery of reductive amination biocatalysts with an enlarged substrate range. This is the first report of wild-type enzymes with related genes having proper NAD(P)H-AmDH activity.

Full text of DOI:10.1039/c6cy01625a

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and C. Vergne Vaxelaire, Catal. Sci. Technol., 2016, DOI: 10.1039/C6CY01625A.
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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Asymmetric reductive amination by a wild-type amine dehydrogenase
from the thermophilic bacteria Petrotoga mobilis
abcϮ
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abc
abc
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abc
Ombeline Mayol, Sylvain David, Ekaterina Darii, Adrien Debard, Aline Mariage, Virginie Pellouin,
abc
abc
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Jean-Louis Petit, Marcel Salanoubat, Véronique de Berardinis, Anne Zaparucha and Carine Vergne-
abc
Vaxelaire.
*
The biocatalytic reductive amination of ketone to chiral amine is one of the most challenging reaction. Using a genome-mining approach,
we found proteins catalyzing the reductive amination of ketones without carboxylic function in α or β position. The synthesis of (4S)-4-
aminopentanoic acid (ee ≥ 99.5%) was achieved with the thermoactive AmDH4 from Petrotoga mobilis in 88 % yield. The high stability and
substrate tolerance make this AmDH4 a very good starting point for further discovery of reductive amination biocatalysts with enlarged
substrate range. This is the first report of wild-type enzymes with related genes having proper NAD(P)H-amine dehydrogenase (AmDH)
activity.
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reduction of various ketones including hydrophobic ones,
through the development of a biphasic aqueous-organic
Introduction
1
5
solvent reaction system. Biocatalytic potential of F-AmDH
was newly highlighted in a remarkable dual-enzyme hydrogen-
Chiral amines are present in many active compounds and are
among the most frequently used chemical intermediates for
1
6
borrowing cascade by Turner and coworkers. The same
cascade has been described by Xu et al. with a double mutant
of leucine dehydrogenase from Exiguobactertium sibiricum
coupled with a low enantioselective alcohol dehydrogenase
the
production
of
agrochemicals,
pharmaceuticals
1
ingredients, polymers and many high value materials. These
secondary or tertiary amines come mostly from the simple
alkylation and/or amidation of chiral primary amines, making
this latter as convenient building blocks adopted by chemists.
Various production procedures have been developed, the
major route being the kinetic resolution by diastereoisomeric
crystallization of racemic amines. Besides, the most two
established chemicals methods are the asymmetric addition of
carbanions to aldimines generated from aldehyde and the
asymmetric hydrogenation of acetamides and imines obtained
from ketones, followed by cleavage of the tertiary amines
1
7
from Streptomyces coelicolor. Very recently, a phenylalanine
dehydrogenase from Rhodococcus sp. M4 was engineered into
an amine dehydrogenase enabling the highly enantioselective
reductive amination of phenylacetone and 4-phenyl-2-
18
butanone. In addition, substantial promises for imine
reductase family (IREDs) in this asymmetric reductive
amination of ketone with ammonia have also been reported
1
9
over the last two years as illustrated recently by engineered
0
IREDs.
2
2
obtained. None of these methods enable the direct access to
To match the demand in synthetically useful AmDH, enzymes
exhibiting excellent stereoselectivity with related genes are
needed. Among established strategies to provide enzymes
possessing new or improved properties, exploring protein
chiral primary amine from ketone without stepping through a
secondary imine or protecting the amine intermediate.
Moreover, most processes require expensive and
unsustainable transition metal complexes and generate
copious amount of waste via protection and deprotection
steps. Thanks to significant advances in genomics, molecular
biology and bioinformatics, high number of natural or redesign
2
1
natural diversity is one of the most attractive. Moreover,
having in hands wild-type AmDHs should expend the synthetic
scope of amine dehydrogenases and furnish new scaffolds for
semi-rational protein engineering.
3
enzymes are now available to the synthetic organic chemist,
making biocatalysis an increasingly attractive manufacturing
5
4
option. At industrial scale, the ω-transaminases, catalyzing
the synthesis of chiral amines from pro chiral ketones with a
Experimental
6
sacrificial amine donor, have become the tools of choice.
7
Chemicals, equipment
Enzymatic reductive amination with ammonia of pro chiral
ketones into chiral amines is naturally abundant but restricted
to the α-keto acid substrates which give chiral α-amino acids
by reaction catalyzed by enzymes of the α-amino acid
All reagents were purchased from commercial sources and
used without additional purification. 4-aminopentan-2-one,
pentan-2-one, 4-oxopentanoic acid, hexan-2 -one, 5-
oxohexanoic acid, glucose, glucose-6-phosphate, (S)-2-(5-
fluoro-2,4-dinitrophenylamino)propanamide (FDAA), NADH,
NADPH were purchased from Sigma-Aldrich. 1-fluoro-2,4-
dinitrobenzene (DNFB) was purchased from Acros Organic,
1
, 8
dehydrogenase family.
As far as we know, there are very
few examples of enzymatic reductive amination by wild-type
amine dehydrogenase (AmDH) active towards ketones without
carboxylic acid moiety in α or β position. Itoh and coworkers
described this activity in Streptomyces virginiae on isolated
racemic 4-aminopentanoic acid rac-
acid rac-10 from Enamine. Recycling enzyme glucose-6-
dehydrogenase (G6PDH) G5885 from Leuconostoc
9 and 5-aminohexanoic
9
enzyme but with low enantioselectivity, and Wang et al. on
1
0
whole-cells of Pseudomonas kilonensis. But until today, no
gene sequence has been identified to encode for these amine
dehydrogenases. Facing the importance of this reaction
mesenteroides and G7877 from S. cerevisiae, and formate
dehydrogenase (FDH) from Candida boidinii were purchased
from Sigma-Aldrich. A sample of phosphate dehydrogenase
1
1
promoted as the holy grail of biocatalysis, hard work has
been done to create such amine dehydrogenase by protein
engineering. Bommarius and coworkers firstly reported the
(
PTDH) was gratefully given by Pr. Marco Fraaije (University of
Groningen). 1g of glucose dehydrogenase (GDH) GDH105 and
CDX901 were given by Codexis®. NMR spectra were recorded
on a Bruker 300 MHz spectrometer (Evry University, France).
1
2
rational design of amine dehydrogenases (L-AmDH and F-
1
AmDH, respectively from Bacillus stearothermophilus and
3
1
13
Chemical shifts (expressed in ppm) of H and C NMR spectra
were referenced to the solvent peaks (H)=3.34 and (C)=49.9
1
Bacillus badius ) using α-amino acid dehydrogenase as
4
δ
δ
starting scaffold. These mutants catalyze the reversible
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for MeOD-d4,
δ
(H)=4.79 for D
TLC) was performed with aluminium-backed sheets with silica 132.0
2
O. Thin-layer chromatography mode
(rac-9:
117.9ꢀ101.0; 117,9
ꢀ
83.1;
rac-10:
(
ꢀ
gel 60 F254 (Merck). Column chromatography was performed UHPLC-UV analyses. UHPLC analyses were performed using a
114.8; 132.0
ꢀ
96.9; 132.0ꢀ69.0).
on a CombiFlash® Companion using Redised® Rf cartridges. Kinetex® F5 (Phenomenex) column (100 x 2.1 mm; 1.7 μm).
UHPLC analyses were performed on a UHPLC U3000 RS 1034 Conditions A: mixture of MeCN/H O + 0.1% formic acid as
2
bar (Thermo Fisher Scientific, Waltham, USA) equipped with
diode array detector DAD3000. HPLC-MS analyses were
performed on a U3000 RS 1034 bar (Thermo Fisher Scientific)
coupled to a Hybrid triple quadrupole-linear ion trap mass-
spectrometer (QTRAP 5500 from ABSciex, Toronto, Canada).
Ion exchange column purification was performed on a
Dowex®50WX8-200 mesh purchased from Sigma Aldrich.
Polarimetry measurements were done at 589 nm on a Unipol L
Polarimeter (Schmidt + Haensch) equipped with a glass tube
eluent with a linear gradient (ratio 30/70 during 2 min, then
0/70 to 80/20 in 2.5 min, then 80/20 during 0.5 min); flow 0.5
3
-1
mL.min ; temperature 25 °C; injection volume 1 μL; UV
detection at λ = 360 nm. Conditions B: mixture of MeCN/H
0.1% formic acid as eluent with a linear gradient (ratio 10/90
during 2 min, then 10/90 to 50/50 in 5 min, then 50/50 to
2
O
+
-1
1
00/0 during 2 min); flow 0.5 mL.min ; temperature 25 °C;
injection volume 1 μL; UV detection at λ = 340 nm.
50 mm.
Library selection for activity
Enzymes preparation
Enzymatic screening assay. All the reactions were conducted
at 25°C in 96-microwell plates. Amination reactions: to a
reaction mixture (100 µL) containing 46 mM of ketone
substrate, 0.5 mM of NADH and 0.5 mM of NADPH in 225 mM
Choice of candidate enzymes. The collection of candidate
2
2, 23
enzymes was built up as previously described,
protein sequence of (2R,4S)-2,4-diaminopentanoate
dehydrogenase (2,4-DAPDH) from Clostridium sticklandii
Uniprot entry: E3PY99, related gene ord).
using the
4 4
NH Cl/NH OH buffer (pH 9.8) was added 30 µL of cell free
extract. Deamination reactions: to a reaction mixture (100 µL)
containing 46 mM of ketone substrate, 0.5 mM of NAD and 0.5
(
Cloning and candidate enzyme production. All steps from
3 2 3
mM of NADP in 100 mM NaHCO /Na CO buffer (pH 9.8) was
primers purchase to cell lysate preparations were carried out
2
as previously described. 20 enzymes were obtained in a 96-
3
added 30 µL of cell free extract. Absorbance at 340 nm was
measured immediately and monitored for 4 h. A background
plate was established in the same manner but with a mixture
lacking ketone (amine in case of deamination reaction)
substrate. Active enzyme corresponds to a well exhibiting a
higher slope in the reaction well over the background well.
HPLC-MS activity confirmation. To a reaction mixture (50 µL)
containing 10 mM of ketone substrate, 4 mM of NADH and 4
microwell plate as cell free extracts.
Purification of enzymes. The enzymes were purified by loading
the cell free extract (8 mL) onto a Ni-NTA column (QIAGEN),
according to the manufacturer’s instructions. Elution buffer
was 50 mM Tris pH 7.5, 50 mM NaCl, 250 mM imidazole and
1
0 % glycerol. The positive fractions were desalted using
Amicon ultracel-10K (Merck Millipore) with buffer containing
0 mM phosphate pH 7.5, 50 mM NaCl and 10 % glycerol to
4 4
mM of NADPH in 225 mM NH Cl/NH OH buffer (pH 9.8) was
5
added 7.5 µL of cell free extract. The reaction mixture was
stirred at 30°C (500 rpm). After the specified period of time
obtain purified enzymes at 1.7 to 5 mg/ml (yield: 0.6 to 2 %
from crude extract). Large scale purification of AmDH4 was
done by heat treatment: cell free extracts (35 mL) were heated
at 70°C for 20 min. After centrifugation (12000 rpm, 4°C, 20
min), the supernatant containing the purified enzyme was
taken. Protein concentrations were determined by the
Bradford method: no significant loss of material (yield: 42%)
and activity was observed. The samples were analyzed by SDS-
Page using the Invitrogen Nupage system (See Figure S1 and
S2). The purified proteins were stored at -80 °C.
(
2h or 3 days), an aliquot (4 µL) was diluted in 396 µL of HPLC
mobile phase (2/8 (NH ) CO 10 mM / MeCN), filtered on 0.22
3
4
2
µm, and analyzed by HPLC-MS following conditions detailed
above. A background reaction was performed in the same
manner but with a mixture lacking the cell free extract. MS-
calibration curves based on MRM responses were established
with commercially available racemic amines rac-9 and rac-10.
Substrates synthesis
HPLC conditions
Synthesis of (2R)-4-oxopentanoïc acid (2). (2R)-4-
oxopentanoïc acid was obtained by hydrogenation followed
2
HPLC-MS analyses. HPLC-MS analyses were conducted on a
by water free acidic treatment of benzyl 2-((tert-
butoxycarbonyl)amino)-4-oxopentanoate synthesized by Alfa
Chemica.
Zic-pHilic (Merck) column (100 x 4.6 mm; 5 μm) with the
following conditions : mixture of (NH
4 2 3
) CO 10 mM / MeCN as
eluent with a linear gradient (ratio 20/80 to 60/40 in 10 min,
-1
Synthesis of 2-amino-5-oxohexanoic acid (rac-3). To a solution
of tert-butyl 2-((diphenylmethylene)amino)acetate (1.63
mmol, 1.1 equiv., 482 mg) in dried THF (8 mL) was added but-
then 60/40 during 6 min); flow 0.5 mL.min ; temperature
4
0°C; injection volume 10 µL. MS parameters: ion source
500V, curtain gas 20 a.u., temperature 450 °C, gas1 45 a. u.,
5
3
-en-2-one (1.48 mmol, 1 equiv., 120 µL) followed by cesium
gas2 60 a.u., CAD – medium; MRM (multiple reaction
monitoring) scan type using multiple ion transitions in positive
carbonate (1.48 mmol, 1 equiv., 482 mg). The mixture was
stirred under inert atmosphere for 16 h at room temperature
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and then concentrated under reduced pressure to remove 400 rpm. After a specified period of time, an aliquot (10 μL)
THF. The residue was then taken up in a mixture of water and was subjected to DNFB derivatization for conversion studies
ethyl acetate and extracted with ethyl acetate (2 x 20 mL). The (see example in Figure S3) or FDAA derivatization for ee
combined organic layers were washed with brine (10 mL), determination. The reactions were compared to blank
dried over magnesium sulfate, filtered and concentrated to reactions performed without enzyme and replicated when
dryness under reduced pressure. Purification of the product by specified. Conversions were deduced from calibration curves
flash chromatography on silica gel (isocratic mode: petroleum obtained with commercial racemic amine.
ether/ethyl acetate 90/10) afforded the desired keto ester as a Procedure for the derivatization with 1-fluoro-2,4-
colorless oil; yield: 500 mg (92 %). The ester (0.24 mmol, 1 dinitrobenzene (DNFB). To 10 µL of a 10 mM reaction mixture
equiv., 86 mg) was treated with 4M hydrochloric acid in were added 10 µL of a 1 M Na
2 3
CO aqueous solution, 50 µL of
dioxane (200 µL) at room temperature and concentrated to a 1 M NaHCO aqueous solution and 40 µL of a 5 mM DNFB
3
dryness under reduced pressure. After trituration (x 3) with solution in EtOH. The mixture was stirred (400 rpm) at 65°C for
dichloromethane, the resulting solid was dried under reduced 1h and then quenched by 50 µL of a 1 M HCl aqueous solution,
pressure to afford the desired 2-amino-5-oxohexanoic acid as filtered (0.22 µm), and analyzed by UHPLC (condition A).
1
hydrochloride salt; yield: 20 mg (48%). H NMR (MeOD-d4, 300 Procedure for the derivatization with (S)-2-(5-fluoro-2,4-
MHz): δ = 5.18 (m, 1H), 3.24 (m, 2H), 2.72 (m, 1H), 2.58 (d, J = dinitrophenylamino)propanamide (FDAA). To 20 µL of a 10
1
.59 Hz, 3H), 2.44 (m, 1H); C NMR (MeOD-d4, 75 MHz): δ = mM reaction mixture were added 8 µL of a 1M NAHCO3
3
1
1
99.6, 171.8, 69.2, 40.2, 26.3, 19.4) (See Figure S6 and S7).
aqueous solution and 20 µL of a 15 mM FDAA solution in EtOH.
The mixture was stirred (400 rpm) at 50°C for1h and then
quenched by 4 µL of 4 M HCl aqueous solution and centrifuged
Activity measurements
(
10 min, 10000 rpm) to remove proteins. The resulting
Specific activities of the selected enzyme. To a mixture of
supernatant (25 µL) was diluted in MeCN (75 µL), filtered (0.22
µm), and analyzed by UHPLC (condition B).
ketone (100 mM), ammonia buffer (300 mM NH Cl/NH OH pH
4
4
7.2) and NADH (0.2 mM) (or NADPH 0.2 mM if the activity was
better in the tested conditions) in a non-thermostated
spectrophotometric cell was added an appropriate amount of
Biocatalytic synthesis of (4S)-4-aminopentanoic acid
enzyme AmDH (reaction final volume 100 µL). The initial slope In a Greiner tube (50 mL) with a screw cap, was poured 9 mL of
measured at 340 nm determined the specific activity of the a media containing 5 M NH CO H/NH Cl buffer (pH 8.5), 0.5 M
4
2
4
enzyme according to Beer-Lambert’s law and molar 4-oxopentanoic acid (6) (basified at pH 8.9), 0.4 mM NADH, 0.5
-
1
-1 24
absorptivity of β-NADH (ɛ = 6,298 M cm ).
U of FDH and 9 mg of AmDH4 purified by heat treatment. The
reaction mixture was stirred at 400 rpm at 50°C for 24 h then
quenched with 6 M HCl aqueous solution to reach pH 1,
centrifuged (10 krpm, 20 min). The supernatant was taken and
the pellet was dissolved in water and centrifuged (10 krpm, 20
min) a second time. Supernatants were combined and
lyophilized overnight. The freeze-dried product was dissolved
in 0.1 M HCl aqueous solution and purified by ion exchange
chromatography using a Dowex® 50WX8-200 mesh resin
previously activated by hydrochloric acid 1M (non-linear
Activity versus temperature. A reaction mixture of ketone
6
(
100 mM) in ammonia buffer (300 mM NH Cl/NH OH pH 7.2)
4 4
heated to the specified temperature in a ThermoMixer®
Eppendorf was added to a thermostated spectrophotometric
cell containing NADH (0.2 mM) and the enzyme AmDH4
purified by heat treatment (reaction final volume 100 µL). The
mixture was rapidly homogenized and the initial slope
measured at 340 nm in a temperature controlled UV-
spectrophotometer set to the specified temperature. The
difference in slope with NADH oxidation with no enzyme at the
same temperature determined the specific activity of AmDH4.
Thermostability studies. To a reaction medium containing a
solution of a 100 mM solution of 4-oxopentanoic acid (6) in
gradient elution (4 column volumes): 0.1 M HCl, H
2
O until
neutral pH, and NH OH 3.3%). Positive fractions (TLC,
4
ninhydrine relevation) were combined and lyophilized to
afford (4S)-4-aminopentanoic acid as a white solid (466 mg,
8
8% yield) with ee ≥ 99.5 % as determined by UHPLC after
FDAA derivatization (Figure S5). tR ( ) =2.30 min (condition A),
tR [(S)- ] = 4.79 min, tR [(R)- ] = 5.09 min (conditions B),
= -11.3° (c = 30,1 mg/ml, H O) {lit. for opposite
300 mM ammonia buffer (NH
4 4
Cl/NH OH pH 7.2) preheated at
9
6
0°C during 5 min, was added a solution of 0.2 mM NADH
9
9
1
5.4
following by 0.5 mg/mL of AmDH4 purified by heat treatment [α]D
previously incubated for specified time at specified enantiomer (4R)-4-aminopentanoic acid: [α]
2
2
2.0
D
= +13.6° (c =
O) δ = 3.34 – 3.19 (m, 1H),
2.20 (ddd, J = 7.8, 7.1, 3.1 Hz, 2H), 1.91 – 1.60 (m, 2H), 1.20 (d,
2
5
1
2 2
temperature. Residual activity was measured at 60°C at 340 1.05, H O) }, H NMR (300 MHz, D
nm as described above.
2
5
13
J = 6.6 Hz, 3H) {identical to literature }; C NMR (75 MHz,
O) δ = 181.9, 47.9, 33.8, 30.9, 17.7 (See Figure S8 and S9).
D
2
Enantioselectivity and conversion
The reactions were performed in a 100 μL volume, containing
substrate 4-oxopentanoic acid (6), specified cosubstrate,
Results and discussion
cofactor recycling enzyme, cofactor NADH, specified buffer,
and specified amount of AmDH at specified temperature and
Identifying natural Amide dehydrogenase (AmDH)
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2
3, 32
To track genes coding for AmDHs among the natural E3PY99) was used in a sequence-driven approach
to build
prokaryotic biodiversity, we first inventory all the reactions a collection of 2,4-DAP dehydrogenase similar enzymes.
transforming unfunctionalized ketones into amines with 169 proteins sharing at least 30 % identity over at least 80 % of
ammonia using NAD(P)H as cofactor, by exploring metabolic the length with E3PY99 were collected. Proteins have been
2
6
databases. All the reactions on α-keto acids were excluded clusterized based on sequence identity (≥ 95%) and one
from the request, removing so any α-amino acid representant per cluster, for which genomic DNA was available
dehydrogenase from the search. We identified L-erythro-3,5 in Genoscope strain collection, was chosen. From the 26
diaminohexanoate dehydrogenase (3,5-DAHDH) involved in candidates selected to be representative of their sequence
2
7
lysine fermentation pathway
and the (2R,4S)-2,4- diversity, 20 genes were successfully cloned in an expression
diaminopentanoate dehydrogenase (2,4-DAPDH) involved in vector. PCR failure occurred for the remaining 6 genes for
2
8, 29
ornithine-fermenting bacteria
assumed to be also active which GC content (<30 % or >65 %) prevent efficient gene
3
0
towards 2,5-diaminohexanoate DAH (Scheme 1).
amplification. After overexpression of recombinant genes in
Escherichia coli strain BL21, cells were lyzed and the protein
quantified (see Supporting Information). These enzymes were
then screened as cell-free extracts against various substrates.
To approach proper amine dehydrogenase substrates, a set of
structural variants of the substrates of 2,4-DAPDH (2 and 3)
with deletion of the amino and/or carboxylic moiety have been
considered (Fig. 1). AmDH activity was assayed in the forward
(reductive amination) or reverse (oxidative deamination)
direction by spectrophotometric NAD(P)H-monitoring or HPLC-
MS (Table 1).
Scheme 1 Reactions catalyzed by L-erythro-3,5 diaminohexanoate DH
and 2,4-diaminopentanoate DH
We decided to focus our attention on enzymes active towards
ketones without α or β carboxylic acid moiety, all the more so
due to the synthesis of various β-amino acids recently
reported by Zhu et al. with variants of 3,5-DAHDH from
Candidatus Cloacamonas acidaminovorans, a bacterium found
3
1
Fig. 1 Compounds selected for the screening of AmDH candidates
in an anaerobic digesters. The protein sequence of 2,4-
DAPDH from Clostridium sticklandii (UniprotKB identifier:
Table 1 Spectrophotometric and HPLC-MS screening assay results
Entry
b
b
Name
UniprotKB
identifier
Organism
Activity
towards
rac-2
Activity towards 6
Activity towards 8
a
c
d
UV
+
UV
nd
+
MS
UV
nd
nd
nd
nd
nd
nd
+
MS
e
1
AmDH1
AmDH2
AmDH3
AmDH4
AmDH5
AmDH6
AmDH7
AmDH8
AmDH9
A6LLG5
A7HNJ8
A8MGL6
A9BHL2
E3PY99
B8D005
D2Z5Z0
D7DCF5
D9RY14
Thermosipho melanesiensis
e
2
3
4
5
6
7
8
9
Fervidobacterium nodosum
Alkaliphilus oremlandii
+
+++
nd
+
nd
+
e
Petrotoga mobilis
+
+++
+++
++
++
Clostridium sticklandii
+
+
e
Halothermothrix orenii
+
nd
+
Dethiosulfovibrio peptidovorans
+
+++
+++
++
e
Staphylothermus hellenicus
+
+
+
++
e
Thermosediminibacter oceani
+
+
+
nd
a
0.01 mM of product; nd: not detected; empty cells: not tested; tested
+: detected; ++: detection of
≥
0.001 mM of product; +++: detection of
≥
b
in oxidative deamination reaction; tested in reductive amination reaction; MS detection of 4-aminopentanoic acid
c
d
after 2h; MS detection of
9
e
-aminohexanoic acid 10 after 3 days; thermophilic organism.
5
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Three compounds were found to be substrates. Nine enzymes one with NADPH. The enzyme seems to have a narrow
demonstrated activity toward ketone , the natural substrate substrate scope as its activity drop to 5.6 mU/mg of enzyme
of 2,4-DAPDH. More interestingly, six of them were found towards the homolog 5-oxohexanoic acid. No activity could be
active towards the 4-oxopentanoic acid ( ), which was detected on acetophenone, cyclohexanone, penta-2-one or 2-
confirmed by MS analysis (Table 1, entries 2, 4, 5, 7-9), and aminopentan-2-one. A profile of temperature versus activity
among the latter three exhibit slight activity towards the towards 4-ketopentanoic acid ( ) revealed that AmDH4 is
2
2
8
6
6
homologous 5-oxohexanoic acid (Table 1, entries 4, 5, 8). Being hardly active at 20 °C and starts to exhibit good activity at 60
active towards non-α/β keto acids, these enzymes can be °C and even above as better activity was found at 90 °C (Fig. 2).
described as the first wild-type ω-amine dehydrogenases.
Its thermal optimum activity could not be reached due to
limitations of our UV-VIS spectrophotometer. At this high
Considering these very promising results, the six enzymes temperature, its specific activity was 8 fold higher than at
AmDH2, AmDH4, AmDH5, AmDH7, AmDH8, AmDH9 cloned 20 °C, reaching an activity of 0,7 U per milligram of protein, a
with a polyhistidine tag were purified by nickel affinity value close to the one of the chimeric cfL1-AmDH (1,7 U mg-1
3
4
chromatography for further studies. Preliminary experiments for p-fluorophenylacetone).
showed a very high enantioselectivity (ee ≥ 95 %) towards
ketone
6 for the six purified enzymes (data not shown). Their
specific activities were determined by measuring the NAD(P)H
absorbance in the reductive amination reaction (Table 2). The
activities on ketone
assumed natural substrate
Among the three enzymes AmDH2, AmDH4 and AmDH5
showing the highest specific activities on , we decided to
6
were 1000 fold lower than the one on
1
(65 U/mg prot. for AmDH5).
2
9
6
focus on AmDH4 from the thermophilic bacteria Petrotoga
mobilis strain DSM 10674. This thermophilic feature usually
offers interesting properties of stabilities for subsequent
3
3
implementation in industrial processes. Moreover, AmDH4
showed activity towards the substrate analogue
AmDH2, suggesting a better substrate promiscuity.
8 unlike
Fig. 2 Profile of activity versus temperature for AmDH4 on 4-
oxopentanoic acid ( ). Reaction conditions: 300 mM NH Cl/NH OH
buffer, pH 7.2, 200 µM NADH, 100 mM . Error bars represent the
6
4
4
Table 2 Specific activity of purified enzymes towards 4-oxopentanoic
acid (6
6
)
standard deviation of three independent experiments.
a
Name
Specific activity (mU/mg prot)
AmDH4
AmDH7
AmDH8
AmDH9
AmDH5
AmDH2
51,9±4.8
2.9±0.1
To estimate the thermostability of AmDH4, the enzyme was
incubated at various temperatures (30 °C, 50 °C, 60 °C, 70 °C
and 80 °C) during different periods of time and the residual
2.9±0.6
3.1±0.2
activity towards compound
half-life of the enzyme was estimated to be 65 h at 60 °C and
0 min at 80 °C. Most notably, this enzyme was stable several
6 was assayed at 60 °C (Fig. 3). The
34.1±2.6
70.9±15.6
3
a
days at 30 °C as its activity was still around 90 % of the initial
activity after 14 days (data not shown).
Reaction conditions: 300 mM NH
NAD(P)H, 100 mM
4 4
Cl/NH OH buffer, pH 7.2, 200 µM
at ambient temperature (triplicate).
6
Temperature studies with AmDH4
The enzyme AmDH4 was easily purified by heat treatment
without notable loss of material and activity. Its coenzyme
specificity was examined by measuring enzymatic activity
towards ketone
6 and either NADH or NADPH as the
coenzyme. The highest activity in tested conditions was
obtained with NADH with an activity 10 times higher than the
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3
6
including synthetic nicotinamide mimic, are required to
increase the benefit of the thermoactivity of the selected
enzyme. Improvements with the formate/FDH recycling
2 3 3 4 3
system regarding buffer (Na CO /NaHCO , NH Cl/NH or
NH HCO ), pH and buffer/ammonia concentration revealed
4
2
optimum conditions at a pH of 8.5 in 5 M ammonium formate,
with the advantage for the latter of being both buffer and co-
substrate. Increasing the initial ketone concentration affected
the conversion but still enabled the nearly complete
conversion of 0.5 M of ketone
6 by using a somewhat larger
concentration of enzyme (1 mg/ml) to perform the reaction
over 24 h. The enantiomeric excess determined by UHPLC-UV
after FDAA derivatization was up to 99.5 % as the minor
enantiomer could not be detected (Supporting Information
Figure S5).
Fig. 3 Thermostability of AmDH4. Residual activity measured at 60 °C
at the indicated time points after incubation of AmDH4 at various
temperatures. Reaction conditions: 100 mM ketone
in 300 mM NH Cl/NH OH buffer, pH 7.2 Error bars reflect the variation
6, 200 µM NADH
4
4
of three independent experiments.
Conversion with cofactor recycle system
+
NH
4
O
NH2
AmDH4
Conditions to perform the biocatalytic conversion of 4-
ketopentanoic acid (6) to optically pure 4-aminopentanoic acid
OH
OH
O
were optimized step by step and monitored by UHPLC-UV
after DNFB derivatization (Fig. 4). As expected, one equivalent
of NADH did not allow complete conversion due to
thermodynamic reaction equilibrium even at the optimized
temperature of 50 °C, where a conversion of around 40 % has
NAD+
O
NADH
6
(
S)-9
CO
2
HCO
-
2
conversion 90 %
isolated yield 88 %
ee > 99.5 %
FDH
nonetheless been reached. To drive the reaction to completion Scheme 2 Synthesis of (4S)-4-aminopentanoic acid ((S)-9) catalyzed by
without providing large excess of expensive coenzyme, AmDH4 with FDH cofactor regeneration system.
35
different NADH-recycling systems (phosphite/PTDH, glucose-
-phosphate/G6PDH, glucose/GDH, formate/FDH) were tested Semi-preparative scale
6
at various temperatures, revealing glucose/GDH and
formate/FDH as the best systems with an optimum
temperature of 60 °C and 50 °C respectively (Supporting
Information Figure S4). Higher temperatures near optimum
temperature of the enzyme activity do not allow obtaining
better conversions due to denaturation of the enzyme of the
recycling system and / or start of impaired cofactor stability.
Thermostable systems for the regeneration of cofactor,
4
63 mg (88 % yield) of the amine
9 were isolated from a 9 mL /
0.5 M-scale reductive amination reaction after simple
purification on strong acidic cation resin Dowex
0WX8®(Scheme 2). Its structure was confirmed by NMR
5
analysis and its stereochemistry determined by polarimetry to
be (S), as is the case for the metabolic substrate of 2,4 DAPDH.
a
Fig. 4 Biocatalytic reaction improvement.
c
6
10 mM, NADH 10 mM, NH
4
Cl 200 mM, AmDH4 0.1 mg/mL, NaHCO
3
/Na
2 3
CO buffer 100 mM pH 9.5,
b
0 °C. idem as conditions a at 50 °C.
3
6
10 mM, NADH 0.4 mM, NH
4
Cl 200 mM, AmDH4 0.1 mg/mL, NaHCO
3
/Na
2
CO
3
buffer 100 mM pH 9.5,
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d
HCOONa 20 mM, FDH 3 U/mL, 50 °C.
e
6
10 mM, NADH 0.4 mM, AmDH4 0.1 mg/mL, NH
4
CO
2
H/NH
4
OH buffer 200 mM pH 8.5, FDH 3 U/mL, 50 °C.
f
idem as conditions d with 5 M buffer. idem as conditions e with substrate
g
0.5 M. idem as conditions f with AmDH4 1 mg/mL. idem as
h
6
conditions g with FDH 0.5 U/mL. Acidic substrat 6 was previously basified at studied pH. All reactions were performed on 100 µL reaction volume
at 400 rpm in 24h.
4
A. S. Bommarius, Ann. Rev. Chem. Bio. Eng., 2015,
6, 319-
3
45; B. M. Nestl, B. A. Nebel and B. Hauer, Curr. Opin. Chem.
Conclusions
Biol., 2011, 15, 187-193; M. T. Reetz, J. Am. Chem. Soc.
2013, 135, 12480-12496; N. J. Turner and E. O'Reilly, Nat.
In summary, NAD(P)H-proper amine dehydrogenase have been
identified among the biodiversity and particularly within the
thermophilic bacteria Petrotoga mobilis strain DSM 10674.
From the latter, AmDH4 was found active towards the 4-
oxopentanoic acid, a γ-ketocarboxylic acid. Optimization of the
reaction conditions including temperature and cofactor
regeneration system enable the sustainable high concentrated
production of (4S)-4-aminopentanoic acid with high yield (88
Chem. Biol., 2013,
9, 285-288; A. S. Wells, G. L. Finch, P. C.
Michels and J. W. Wong, Org. Proc. Res. Dev., 2012, 16
,
1986-1993; R. Wohlgemuth, Curr. Opin. Biotechnol., 2010,
21, 713-724.
5
6
7
J. M. Choi, S. S. Han and H. S. Kim, Biotechnol. Adv., 2015,
33, 1443-1454.
H. Kohls, F. Steffen-Munsberg and M. Hohne, Curr. Opin.
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%) and excellent enantiomeric excess (> 99.5 %). The robust
and easily purifiable enzyme AmDH4 offers a simple access to
this valuable optically pure primary amine, already used as
E. O'Reilly and N. J. Turner, Persp. Science, 2015, 4, 55-61; N.
J. Turner and M. D. Truppo, in Sustainable Catalysis, John
2
5, 37
building block for the synthesis of therapeutic molecule.
Wiley & Sons, Inc., 2013, pp. 63-74.
This is the first example of a wild-type enzyme with related
gene catalyzing the reductive amination of ketone without
carboxylic acid moiety in α or β positions. Its robustness makes
it a good scaffold for directed evolution, offering significant
8
9
M. Höhne and U. T. Bornscheuer, ChemCatChem, 2009, 1,
42-51; J. H. Schrittwieser, S. Velikogne and W. Kroutil, Adv.
Synth. Catal., 2015, 357, 1655-1685.
N. Itoh, A. Matsuyama, Y. Kobayashi, 2002, EP1020515 A2;
3
8
potential for enzyme improvement. Facing the importance of
such reaction in chiral amine synthesis, this discovery will
N. Itoh, C. Yachi, T. Kudome, J. Mol. Catal. B: Enz., 2000, 10,
281-290.
certainly contribute to expending the synthetic scope of amine 10 S. Wang, B. Fang, 2013, CN103224963 A.
dehydrogenase-catalyzed reductive amination in industry. 11 R. A. Sheldon, Multi-step Enz. Catal., Wiley Online Library,
Other natural enzymes with broader substrate range are
2008, 109-135.
currently sought in our laboratory to expand this unique class 12 A. S. Bommarius, M. J. Abrahamson, B. Bommarius, 2014,
of biocatalysts for the synthesis of primary chiral amines.
US2013309734 A1.
1
1
3
4
M. J. Abrahamson, E. Vasquez-Figueroa, N. B. Woodall, J. C.
Moore, A. S. Bommarius, Ang. Chem. Int. Ed. Engl. 2012, 51
,
3
969-3972.
Acknowledgements
M. J. Abrahamson, J. W. Wong and A. S. Bommarius, Adv.
Synth. Catal., 2013, 355, 1780-1786.
We are grateful to Pr. Marco Fraaije for giving us a sample of
optimized phosphite dehydrogenase and Codexis® for sending 15 S. K. Au, B. R. Bommarius and A. S. Bommarius, ACS Catal.,
us free of charge one gram of glucose dehydrogenase GDH-
2014, , 4021-4026.
05 and CDX-901. We thank A. Perret for helpful discussions 16 F. G. Mutti, T. Knaus, N. S. Scrutton, M. Breuer and N. J.
4
1
and useful comments on the manuscript. This work was
Turner, Science, 2015, 349, 1525-1529.
supported by grants from Commissariat à l’énergie atomique 17 F.-F. Chen, Y.-Y. Liu, G.-W. Zheng and J.-H. Xu,
et aux énergies alternatives (CEA), and by a PhD studentship
ChemCatChem, 2015, , 3838-3841.
OM) from the University Paris-Saclay (Evry Val d’Essonne).
18 L. J. Ye, H. H. Toh, Y. Yang, J. P. Adams, R. Snajdrova and Z.
Li, ACS Catal., 2015, , 1119-1122.
7
(
5
1
9
G. Grogan and N. J. Turner, Chem. Eur. J., 2015, 21, 1-9; P. N.
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Catalysis Science & Technology
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DOI: 10.1039/C6CY01625A
Biocatalytic potential of a new wild type amine dehydrogenase used in an enzyme-catalyzed synthesis of an
enantiomerically pure primary amine