Amine Transaminase from Exophiala Xenobiotica - Crystal Structure and Engineering of a Fold IV Transaminase that Naturally Converts Biaryl Ketones

Article abstract of DOI:10.1021/acscatal.8b04524

Amine transaminases are frequently used for the production of chiral amines starting from prochiral ketones. These amines can be applied as active pharmaceutical ingredients or drug precursors. However, there are still limitations to the use of amine transaminases when it comes to bulky ketone substrates, such as biaryl ketones. Using data mining, an (R)-selective amine transaminase from Exophiala xenobiotica was identified which naturally converts biaryl ketone substrates to the corresponding amines with up to 85% conversion and excellent enantioselectivity (>99% ee). Its protein crystal structure was obtained with a resolution of 1.52 ?, which enabled us to explain this interesting substrate acceptance. Structure-guided protein engineering resulted in a quintuple variant with increased stability. Moreover, the amino acid exchange T273S increased the activity and broadened the substrate scope, enabling conversions of various biaryl ketones with up to >99%. A preparative biotransformation of 1-(4-(pyridin-3-yl)phenyl)ethenone at 75 mM (15 g/L) resulted in 96% of isolated yield of the respective amine.

Full text of DOI:10.1021/acscatal.8b04524

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Article
Amine Transaminase from Exophiala xenobiotica – Crystal Structure and
Engineering of a Fold IV Transaminase that Naturally Converts Biaryl Ketones
Aline Telzerow, Juraj Paris, Maria Håkansson, Javier Gonzalez-Sabin, Nicolas Rios-Lombardia, Martin
Schuermann, Harald Gröger, Francisco Morís, Robert Kourist, Helmut Schwab, and Kerstin Steiner
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04524 • Publication Date (Web): 20 Dec 2018
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Amine Transaminase from Exophiala Xenobiotica –
Crystal Structure and Engineering of a Fold IV
Transaminase that Naturally Converts Biaryl
Ketones
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Aline Telzerow,a,b Juraj Paris,c,d Maria Håkansson,e Javier González-Sabín,d Nicolás Ríos-
Lombardía,d Martin Schürmann,b Harald Gröger,c Francisco Morís,d Robert Kourist,a Helmut
Schwaba and Kerstin Steinera,*
[a]
Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14,
8010 Graz, Austria
[b]
[c]
InnoSyn B. V. Urmonderbaan 22, 6167RD Geleen, The Netherlands
Chair of Organic Chemistry I, Faculty of Chemistry, Bielefeld University,
Universitätsstraße 25, 33615 Bielefeld, Germany
[d]
[e]
Entrechem, S.L., Vivero Ciencias de la Salud, Santo Domingo de Guzmán, s/n, 33011
Oviedo, Spain
SARomics Biostructures AB, Medicon Village, Scheelevӓgen 2, 22381 Lund, Sweden
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ABSTRACT: Amine transaminases are frequently used for the production of chiral amines
starting from prochiral ketones. These amines can be applied as active pharmaceutical
ingredients or drug precursors. However, there are still limitations to the use of amine
transaminases when it comes to bulky ketone substrates, such as biaryl ketones. Using data
mining, an (R)-selective amine transaminase from Exophiala xenobiotica was identified which
naturally converts biaryl ketone substrates to the corresponding amines with up to 85%
conversion and excellent enantioselectivity (>99% ee). Its protein crystal structure was obtained
with a resolution of 1.52 Å, which enabled us to explain this interesting substrate acceptance.
Structure-guided protein engineering resulted in a quintuple variant with increased stability.
Moreover, the amino acid exchange T273S increased the activity and broadened the substrate
scope enabling conversions of various biaryl ketones with up to >99%. A preparative
biotransformation of 1-(4-(pyridin-3-yl)phenyl)ethenone at 75 mM (15 g/L) resulted in 96% of
isolated yield of the respective amine.
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KEYWORDS: amine transaminase, biaryls, biosynthesis, enzyme catalysis, protein engineering
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INTRODUCTION
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In the pharmaceutical industry, chiral amines play an important role as active pharmaceutical
ingredients and as building blocks for drugs.[1] When these compounds are produced through
biocatalysis, excellent enantioselectivity and therefore an enantiomeric excess of >99% ee as
required for pharmaceuticals can be obtained. One class of enzymes that is used to produce chiral
amines are amine transaminases (ATAs).[2] These enzymes reversibly catalyze the transfer of an
amine group from an amino donor to a ketone or aldehyde. The co-factor pyridoxal 5’-phosphate
(PLP) plays a crucial role by forming a Schiff base with the active site lysine of the enzyme.[3]
PLP-dependent enzymes are classified according to their fold. ATAs belong to fold classes I and
IV. In contrast to α-amino acid aminotransferases, ATAs are not dependent on an α-carboxylic
acid moiety, therefore not limiting the product range to α-amino acids.[4] Due to advanced
protein engineering of existing biocatalysts or through novel enzyme discovery, the substrate
scope of ATAs has been extended significantly, and several (S)-selective ATAs (fold-class I)
have been engineered to convert a variety of sterically demanding ketones.[5] However, for (R)-
selective ATAs (fold-class IV) the substrate spectrum is less versatile.[6] At the time, when our
project started, only the engineered Arthrobacter ATA-117 from Codexis and Merck & Co,
which was developed for Sitagliptin synthesis, was able to convert highly bulky ketones.[7]
Particularly, the biaryl moiety has received increased attention as a privileged structure by the
pharmaceutical industry, with examples of activity across a wide range of therapeutic substance
classes including antifungal, anti-inflammatory, antirheumatic, antitumor, and antihypertensive
agents.[8] The interest in this scaffold is further evident, as Bornscheuer and coworkers in parallel
to us targeted the (R)-TA from Aspergillus fumigatus by protein engineering to make it suitable
for the conversion of biarylic substrates.[9]
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Our aim was to identify new (R)-selective ATAs that add to the existing toolbox of ATAs and
expand the substrate scope within previously inaccessible bulky substrates. In this study we
report the identification, characterization and enzyme engineering of an (R)-selective amine
transaminase from Exophiala xenobiotica which naturally converts biaryl ketone substrates to
the corresponding amines with up to 85% conversion and excellent enantioselectivity (>99% ee).
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MATERIAL AND METHODS
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Merck KGaA
(Darmstadt, Germany) or Fluorochem (Hadfield, UK), if not stated otherwise. Materials for
molecular biology were obtained from Thermo Fisher Scientific (Waltham, MA, USA), if not
specifically mentioned.
Data Mining and Identification of a Suitable Amine Transaminase
Published fold IV amine transaminase (ATA) sequences[7,10] were used to search for related
sequences in the NCBI database (pBLAST). [11] The obtained protein sequences were aligned by
Clustal with Mega6[12] and hits lacking the motif characteristic for fold IV ATAs[4] (Table S1)
were excluded. The remaining sequences and their host organisms were screened for interesting
features. The habitats of these organisms can for example give an indication on extremophilic
environments resulting in particularly stable proteins or influencing the substrate scope of the
enzymes. The transaminase (XP_013320890) from Exophiala xenobiotica was chosen according
to the origin of its host organism. The black yeast E. xenobiotica was isolated from environments
that were associated with toxic, aromatic xenobiotics such as oil sludge, creosote-treated railway
tie, soil polluted by gasoline or brown coal rich in phenolic compounds.[13] The hypothesis was
that the presence and density of so many different aromatic compounds in the habitat of E.
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xenobiotica might have led to a natural evolution of its amine transaminase to tolerate or accept
these compounds.
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Cloning and Mutagenesis
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The E. xenobiotica ATA gene was ordered codon-optimized for expression in E. coli from
Geneart/LifeTech (Vienna, Austria). The gene was cloned via restriction digestion
(NdeI/HindIII) and ligation (T4 ligase) into pMS470Δ8.[14] To add a C-terminal His-tag for
purification, the transaminase gene of EX-ωTA was sub-cloned via PCR amplification and
restriction digest (NcoI/XhoI) into the expression vector pET28a(+) from Novagen/Merck
(Darmstadt, Germany). Desired mutations were introduced with PCR using primers with the
desired mutation (Table S2) and the Phusion high fidelity polymerase.
Protein Expression and Purification
After transformation of E. coli Top10F’ cells (Invitrogen/LifeTechnologies, Carlsbad, CA, USA)
with pMS470Δ8_EX or E. coli BL21-Gold(DE3) (Stratagene, La Jolla, CA, USA) with
pET28a(+)_EX, the cells were grown at 37 °C in terrific broth medium supplemented with
ampicillin (Amp: 100 µg mL-1) or kanamycin (Kan: 40 µg mg-1). Expression was induced with
0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at an OD600 between 0.6-0.8 and carried
out at 25 °C overnight. The cells were harvested at 4000 x g for 15 min and resuspended in
buffer (pMS470Δ8 constructs: 50 mM KPi buffer, pH 7.5, 0.1 mM PLP; or pET28a(+)
constructs: buffer A: 50 mM MES buffer, pH 6, 0.1 mM PLP, 50 mM NaCl, 10 mM imidazole).
The cells were disrupted by sonication (Branson Sonifier S-250, 6 min, 80% duty cycle, 70%
output) and centrifuged at 50000 x g for 1 h. The cleared lysates were filtered through 0.45 µm
syringe filters. The protein concentration of the lysate was determined by Bradford protein assay.
The expression and solubility of the proteins was analyzed by SDS-PAGE (NuPAGE Bis-Tris
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PreCast Gels/Life Technologies). For purification of the His-tagged protein, the filtered cell-free
lysate was incubated with Ni SepharoseTM 6 Fast Flow resin (GE Healthcare) for 20 min. The Ni
SepharoseTM resin was then filled into empty PD-10 columns (GE Healthcare). After removal of
impurities with buffer A containing 30 mM imidazole, the target protein was eluted with 300
mM imidazole in buffer A. Fractions were analyzed by SDS-PAGE, pooled, concentrated
(Vivaspin 20 Centrifugal Filter Units; 10000 Da molecular weight cut-off; Sartorius, Göttingen,
Germany) and desalted on PD-10 desalting columns (GE Healthcare) into 20 mM MES buffer
pH 6 (containing 0.1 mM PLP, 50 mM NaCl). The concentration of the purified protein was
determined with a Nanodrop spectrophotometer (model 2000c, Peqlab, Erlangen, Germany),
using an absorbance of 1.43 at 280 nm, calculated based on the amino acid sequence using
ProtParam.[15] The protein was frozen and stored at -20 °C until further use.
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Crystallization and Structure Determination
Screening for crystallization conditions was performed with a mosquito robot (TTP Labtech)
using the screens JCSG+ and PACT premierTM (Molecular Dimensions) by the sitting drop
vapor-diffusion method in MRC 3-well plates. A stock solution of purified EX-ωTA at 11 mg
mL-1 in 20 mM MES buffer pH 6 (containing 0.1 mM PLP, 50 mM NaCl) was used for all
crystallization experiments. Drops of 400 nL were pipetted for screens with a 1:1 ratio of protein
and screening solution. The crystallization plates were incubated in the dark at room
temperature. Initial crystals of EX-ωTA were obtained at several crystallization conditions. A
seed solution was made using seed beads (Hampton Research) and a mixture of initial crystals
from several conditions of the JCSG+ screen stabilized in 100 mM SPG buffer, pH 8.5 and 25%
(w/v) PEG1500. Seeding experiments were set up using 200 nL protein mixed with 30 nL seed
and 170 nL reservoir. A new crystal form appeared in a condition containing 0.1 M sodium
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acetate, pH 4.7 and 2 M ammonium sulfate. The crystal was transferred to a cryosolution
containing 0.1 M sodium acetate (pH 4.6), 0.17 M ammonium sulfate, 25.5% w/v PEG1500,
15% v/v glycerol and flash-frozen in liquid nitrogen. Data collection was performed on the
synchrotron beamline 14.1 (BESSY, Berlin, Germany) at 100 K. The data sets were processed
and scaled using the XDS program package.[16] Molecular replacement was performed with
Phaser[17] using the structure of the Aspergillus terreus ATA (PDB ID: 4CE5)[10e] as a search
template. The resulting model was manually completed in Coot[18] and refined using Refmac5.[19]
Data collection and processing statistics are summarized in Table S3. The final structure was
checked using MolProbity.[20] The coordinates have been deposited in the protein data bank with
accession code 6FTE. Since size exclusion chromatography suggested that the biological
assembly of EX-ωTA is a dimer, which is in accordance with literature, the dimeric pdb file was
generated with QtPISA. [21]
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Substrate Docking and Visualization of Active Site Electrostatics
Docking studies were performed with YASARA (version 18.4.24). To prepare the structure
6FTE for docking, the anisotropic temperature factors, any waters and other ligands were
removed. The co-factor PLP was kept in the structure. After adding any missing hydrogens, the
YASARA standard protocol for energy minimization was applied to the dimeric EX-ωTA
structure. Structures of the substrates were drawn with ChemDraw (version 17.0.0.206(121)) and
the energy was minimized with YASARA. A simulation cell was created around one active site
of the dimeric structure of EX-ωTA and VINA docking was performed with the standard
mcr_dock with 999 runs. The docking result was chosen according to the best energy but also
manual visualization and realistic positioning of the substrate.
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For the visualization of the active site charge, PDB structures were loaded into PyMOL (version
2.1.1). The active site charge was calculated with the APBS electrostatics plugin for PyMOL and
visualized accordingly.
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Spectrophotometric Activity Assay
The activity of the ATA variants was determined with an adjusted standard photometric assay.[22]
Here, the substrate phenylethylamine is converted to acetophenone, which can be detected
spectrophotometrically at 300 nm. The absorbance increases with rising product concentration.
To measure the activities of the ATA variants, reactions were set up in 1 mL volume with 100
mM KPi buffer pH 7.5 containing 0.1 mM PLP, 5 mM phenylethylamine and 5 mM pyruvic
acid. For the reaction, 50 µg to 250 µg of total protein were used. The production of
acetophenone was measured for several minutes at 300 nm at 30 °C. The volumetric activity in U
mL-1 and the specific activity in U mg-1 total protein of the enzyme samples was calculated using
the Beer-Lambert law with the molar extinction coefficient of acetophenone ε300 = 0.28 cm2
µmol-1.
Biotransformations
Enzymes
LDH (Lactate dehydrogenase) from rabbit muscle (Sigma-Aldrich, Type II, ammonium sulfate
suspension, 800-1, 200 U mg-1 protein).
GDH (Glucose dehydrogenase) from Bacillus megaterium was expressed in Escherichia coli.
The activity was determined as 1884 U mL-1 according to the assay described in literature.[23]
EX-ωTA (amine transaminase from Exophiala xenobiotica) and its variants were expressed in
Escherichia coli. The activity was measured spectrophotometrically (see above) and yielded:
EX-wt: 16.9 U mL-1 or 4.5 U mg-1 total protein, EX-X5: 37.7 U mL-1 or 4 U mg-1 total protein,
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EX-STA: 23.35 U mL-1 or 4.6 U mg-1 total protein and EX-STA5: 23.35 U mL-1 or 1.9 U mg-1
total protein.
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The (S)-alcohol dehydrogenase from Rhodococcus ruber DSM 44541 was provided as a cell-free
extract (CFE) by InnoSyn B.V. (Geleen, The Netherlands).[24] The total protein concentration
was about 46 mg mL-1 with a good expression level of the ADH in the soluble fraction. The
activity was determined spectrophotometrically at pH 6.0 with acetone and NADH as substrates
and was 225 U mL-1 or 4.85 U mg-1 total protein, respectively.
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Reagents
1-(Biphenyl-4-yl)ethanone (1a) was purchased from Merck Schuchardt. Biaryl ketones (1b-j)
were prepared as described in the literature.[25] D-(+)-Glucose was purchased from VWR. D-
Alanine, PLP (Pyridoxal 5′-phosphate hydrate) and NAD+ were purchased from Sigma Aldrich.
General methods
1H-NMR and proton-decoupled 13C-NMR spectra (CDCl3) were obtained using a Bruker DPX-
300 (1H, 300.13 MHz and 13C, 75.5 MHz) spectrometer using the δ scale (ppm) for chemical
shifts. Calibration was made on the signal of the solvent (13C: CDCl3, 77.16; 1H: CDCl3,
7.26).[26] High resolution mass spectra were recorded on a Bruker Impact II instrument. Optical
rotations were measured using a Perkin-Elmer 241 polarimeter and are quoted in units of 10-1deg
cm2 g-1. HPLC analyses to determine the degree of conversion were carried out in an Agilent
RR1200 HPLC system, using a reversed phase column (Zorbax Eclipse XDB-C18, RR, 18 μm,
4.6 x 50 mm, Agilent). HPLC analyses to determine ee values were performed on a Hewlett
Packard 1100 LC liquid chromatograph. Details of HPLC methods and analyses including
chromatograms can be found in the supporting information.
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General Procedure for the Screening of Ketones 1a-r using EX-TA (Wild Type and Variants)
and Alanine as Amino Donor
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The ketone (20 mM) was first dissolved in DMSO (32 μL, 5% v/v) in a 2 mL reaction tube
(Eppendorf). Then EX-TA lysate (1a-j: 8 U, 1k-r: 20 U), potassium phosphate buffer (KPi) 100
mM pH 7.5, 1 mM PLP, 0.1 mM NAD+, D-glucose (57 mM), D-alanine (130 mM), LDH (90 U)
and GDH (30 U) were added. The reaction was shaken at 30 ºC and 250 rpm for 24 h. To determine
the conversion, 10 µL of the mixture were diluted with 90 µL of DMSO and analyzed by achiral
reverse phase with previous centrifuging and filtering of the sample. To determine the
enantiomeric excess for reactions employing the ketones 1a-j, the reaction was quenched by
addition of aqueous 10 N NaOH (400 μL). The mixture was then extracted with ethyl acetate (2 ×
500 µL) and the organic layers were separated by centrifugation (90 s, 13000 rpm), combined and
dried over anhydrous Na2SO4. The enantiomeric excess of amines was measured by chiral HPLC.
General Procedure for the Screening of Biaryl Ketones 1a-j using EX-STA and Isopropylamine
as Amino Donor
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In a 2 mL reaction tube, ketone (20 mM) was first dissolved in DMSO (32 μL, 5% v/v) and then
ATA lysate (8 U), KPi buffer 100 mM pH 7.5, 1 M isopropylamine and 1 mM PLP were added.
The reaction was shaken at 30 ºC and 250 rpm for 24 h. To determine the conversion, 10 µL of the
mixture were diluted with 90 µL DMSO and analyzed by achiral reverse phase with previous
centrifuging and filtering of the sample. To determine the enantiomeric excess the reaction was
quenched by addition of aqueous 10 N NaOH (400 μL). The mixture was then extracted with ethyl
acetate (2 × 500µL) and the organic layers were separated by centrifugation (90 s, 13000 rpm),
combined and dried over anhydrous Na2SO4. The enantiomeric excess of amines was measured by
chiral HPLC.
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General Procedure for the Synthesis of Racemic Amine Standards 2a-g
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Ammonium formate (300 mg) and zinc (150 mg) were added to a solution of 80 mg ketone (1a-
g) in 1.5 mL of ethanol and the mixture was stirred overnight at 80 ºC under nitrogen. Then, the
reaction mixture was cooled at room temperature, filtered through Celite and washed with
ethanol. The solvent was removed under vacuum to obtain an oily crude, followed by the
addition of 10 mL of aqueous 1 N HCl and washing with diethyl ether (2 × 20 mL). The aqueous
layer was basified to pH 10 with aqueous 10 N NaOH and extracted with ethyl acetate (3 × 5
mL). The organic layer was washed with 10 mL of brine and dried over anhydrous Na2SO4.
Further concentration on a rotary evaporator afforded the corresponding biaryl amine 2a-g (25-
30% yield).
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General Procedure for the Synthesis of Racemic Amine Standards 2h-j
Et3N (2 eq) and MsCl (2 eq) were added to a solution of 80 mg alcohol (beforehand obtained by
reducing the corresponding ketone with NaBH4) in 3.0 mL of dichloromethane and the mixture
was stirred overnight at room temperature. Then, the solvent was removed and NaN3 (10 eq) and
anhydrous DMF (3 mL) were added, the resulting mixture being stirred at 70 ºC for 24 h. Further
evaporation of the solvent under reduced pressure afforded a crude which was purified by flash
chromatography (EtOAc/hexane mixtures). Finally, to a suspension of the previous azido
compound and Pd-C 10% (25 mg) in a round-bottom flask, a H2 balloon was connected and
deoxygenated MeOH (1.5 mL) carefully added. The resulting mixture was stirred at room
temperature for 24 h. After this time, the mixture was filtered through Celite® and the solvent
evaporated under reduced pressure to afford the corresponding biaryl amine 2h-j (yield: 70-
75%).
General Procedure for the Synthesis of Enantiopure Biaryl Amine Standards
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In a 15 mL reaction tube, ketone (0.25 mmol) and ADH from R. ruber (16 U) were added to a i-
PrOH (5 mL)/50 mM KPi buffer pH 7.0 (5 mL) mixture (1 mM NAD+). The reaction was shaken
at 30 ºC and 250 rpm for 24 h. The mixture was extracted with ethyl acetate (2 × 10 mL), the
organic layers combined, dried over anhydrous Na2SO4, filtered and concentrated under vacuum
to provide the corresponding (S)-alcohol (ee >99%).
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Then, the corresponding (S)-alcohol (0.25 mmol) and diphenyl phosphoryl azide (DPPA, 0.30
mmol) were dissolved in dry toluene (0.4 mL). The mixture was cooled to 0 ºC under N2, and
DBU (0.30 mmol) was added. The reaction was stirred for 2 h at 0 ºC and then at 20 ºC for 16 h.
The resulting mixture was washed with H2O (2 × 5 mL) and aqueous 1N HCl (5 mL). The
organic layer was concentrated under reduced pressure and purified by flash chromatography
(EtOAc/hexane mixtures). Finally, to a suspension containing the resulting azide compound and
Pd-C 10% (10 mg) in a 25 mL round-bottom flask a H2 balloon was connected and deoxygenated
MeOH (1.0 mL) carefully added. The resulting suspension was stirred at room temperature for
24 h. After this time, the reaction mixture was filtered through Celite® and the solvent
evaporated under reduced pressure to afford the corresponding (R)-biaryl amine as a colorless oil
(ee = 97%, yield = 50-70%).
Preparative-scale Transamination of 1i using Lyophilized EX-STA
After dissolving 1i (75 mM, 100 mg) in DMSO (319 μL, 5% v/v) in a 50 mL reaction tube,
lyophilized EX-STA (320 U, 700 mg), 100 mM KPi buffer pH 7.5 (5.8 mL, 1 mM PLP, 0.1 mM
NAD+), D-glucose (114 mM, 293 mg), D-alanine (260 mM, 332 mg), LDH (3604 U, 682 μL) and
GDH (1201 U, 400 mg) were added. The reaction was shaken at 30 ºC and 250 rpm for 24 h. The
reaction was quenched by addition of aqueous 10 N NaOH (10 mL) to adjust the pH to 14. The
mixture was then extracted with ethyl acetate (2 × 20 mL) and the organic layers were separated
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by centrifugation (90 s, 13000 rpm), combined and dried over anhydrous Na2SO4. Further
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evaporation under reduced pressure provided (R)-2i as a white solid in 96% yield (ee >99%).
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RESULTS AND DISCUSSION
To identify new (R)-selective ATAs that expand the substrate scope within previously
inaccessible bulky substrates several sequences from literature were used for sequence-based
similarity searches against the NCBI database.[7,10] (R)-amine transaminases are members of the
fold type IV PLP-dependent enzyme family.[7,10] This family includes D-amino acid
aminotransferases, L-branched chain aminotransferases and 4-amino-4-deoxychorismate lyases.
In an analysis of fold class IV enzymes Bornscheuer and his group showed that amino acids in
the active site, which are important for substrate binding, are conserved. Thus, they proposed
motifs that can be used for the prediction of subgroups within this fold class (Table S1). [4]
Sequences that lacked the motif characteristic for ATAs[4] were removed from the pool.
EX-TA converts biarylic ketones
Exophiala xenobiotica-ATA (EX-TA) was chosen based on the habitat of its host organism.
The black yeast E. xenobiotica was isolated from environments that were associated with toxic,
aromatic xenobiotics such as oil sludge, creosote-treated railway tie, soil polluted by gasoline or
brown coal rich in phenolic compounds.[13] The hypothesis was that the presence and density of
so many different aromatic compounds in the habitat of E. xenobiotica might have led to a
natural evolution of its transaminase to accept some substrates that other ATAs do not tolerate.
With these premises, the bioamination of 4’-phenylacetophenone (1a) catalyzed by EX-TA
(Scheme 1) was investigated as a benchmark reaction.
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NH2
O
R
EX-
TA/ PLP
D-Ala, LDH/GDH system

Ar
R
Ar
KPi buffer pH 7.5, DMSO
30 ºC, 250 rpm
2a-j
1a-j
O
O
O
O
9
1a
1b
1d
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1c
O
O
O
OMe
F
F
F
1f
1e
1g
F
O
O
O
N
N
1i
N
1j
1h
Scheme 1. Amine transaminase-catalyzed transamination of biaryl ketones 1a-j employing EX-
TA.
Initially, the reaction was tested under the simplest reaction conditions, namely using
isopropylamine (IPA) as amino donor. Indeed, this reagent is the preferred amino donor in
industry due to its low cost and easy removal of the by-product acetone to shift the
equilibrium.[27] However, despite testing excess of IPA at several donor-acceptor ratios in a
broad pH range, no conversion was detected in any case. In fact, many wild-type ATAs are
reluctant to accept IPA and this problem is only overcome after protein engineering. Thus,
subsequently alanine was used as amino donor combined with the LDH/GDH system to shift the
equilibrium. After thorough parametrization encompassing not only the involved enzymes and
cofactors but also pH, amino donor and co-solvent, the enzymatic system worked efficiently
according to the following reaction setup: 1a (20 mM) in 100 mM phosphate buffer pH 7.5,
supplemented with PLP (1 mM), NAD+ (0.1 mM), glucose (60 mM), D-alanine (6.5-fold) and
DMSO (5% v/v). 8 U of EX-TA were used (Scheme 1). After 24 h of incubation at 30 ºC and
250 rpm, the corresponding biaryl amine (R)-2a was produced enantiomerically pure (>99% ee,
Table 1, entry 1) with a conversion of 83%.
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Table 1. Substrate scope of EX-TA and its variants. EX-STA variant represents amino acid
exchange in the small binding pocket. EX-5 was engineered for improved stability. The variant
EX-STA5 inherits the combined amino acid exchanges of EX-STA and EX-5.[a]
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EX-
wt
EX-
EX-
Entry Substrate
EX-5
ee (%)[b]
STA
STA5
1
2
1a
1b
1c
1d
1e
1f
83
38
-
45
85
-
15
28
-
45
85
-
>99 (R)
>99 (R)
n.d.[c]
3
4
-
-
-
-
n.d.[c]
5
25
21
25
72
82
85
72
40
30
>99
>99
>99
84
30
30
40
50
82
55
30
24
80
95
70
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
6
7
1g
1h
1i
8
9
10
1j
[a] Reaction conditions: 20 mM ketone dissolved in DMSO (5% v/v), 100 mM potassium
phosphate buffer, pH 7.5, 1 mM PLP, 0.1 mM NAD+, cell free E. coli lysate containing 8 U
ATA (according to a spectrophotometric assay at 30°C in 100 mM potassium phosphate buffer,
pH 7.5, 0.1 mM PLP, 5 mM phenylethylamine and 5 mM pyruvic acid as substrates), 130 mM
D-alanine, 60 mM glucose, 30 U GDH and 90 U LDH, shaken for 24 h at 250 rpm and 30 ºC.
Conversions were measured by HPLC. [b] Measured by chiral HPLC; the ee is the same for all
four enzymes. [c] Not detected due to low activity.
Encouraged by this result, the substrate scope of EX-TA was explored employing a panel of
ortho-, meta-, para-biaryl and arylpyridine ketones, some of them exhibiting different patterns of
substitution at the phenyl moiety next to the carbonyl group (Table 1, entries 2-10). While the
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enzyme showed no detectable catalysis toward the ortho-biaryl ketone 1c (Table 1, entry 3), a
remarkable activity was observed for meta- and para-biaryl ketones. In particular, the
unsubstituted arylpyridine ketones 1h-j rendered conversions higher than 70% (Table 1, entries
8-10). The non-conversion toward 4’-phenylpropiophenone (1d) unveiled the size restrictions of
EX-TA in the small binding pocket, which cannot accommodate substituents larger than a
methyl group. Fluorine atoms on different positions on the aromatic ring in ketones 1e-g had a
negative impact on the conversion which was attributed to the strong electron-withdrawing effect
of fluorine (Table 1, entries 5-7). It is worth noting that EX-TA displayed excellent asymmetric
induction in the reductive amination of the carbonyl group of all the ketones, leading to the (R)-
enantiomer of each amine with >99% ee.
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Structural characterization of EX-TA
For a better understanding of this unique substrate acceptance, the crystal structure of EX-TA
was determined by X-ray crystallography at a resolution of 1.52 Å (PDB ID: 6FTE, Figure 1A,
Table S3). The asymmetric unit contains one polypeptide chain. Size exclusion chromatography
suggested that the biological assembly of EX-ωTA is a dimer, which is in accordance with
literature.[10] The homo-dimeric structure of EX-TA showed the typical fold IV with the active
site at the interface of the monomers and the co-factor PLP bound to the active site lysine 179
(Figure 1B). The electron density in the active site was well defined where the PLP was expected
to bind (Figure S1). Moreover, additional density corresponding to a putative glycerol molecule
was observed in the active site. EX-TA also contains an N-terminal α-helix typical for (R)-
selective amine transaminases[10], which is not observed in any of the other fold IV subfamilies.
A structural similarity search using the DALI server[28] confirmed that the closest homologous
structures are indeed (R)-selective transaminases (PDB-code: 4CE5 from Aspergillus terreus[10e]
,
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Z-score: 51.1, rmsd 0.7 Å, seq id: 75%; PDB-code: 4CMD from Nectria haematococca [10b], Z-
score: 50.6, rmsd 0.7 Å, seq id: 74%; PDB-code: 4CHI from Aspergillus fumigatus[10a], Z-score:
50.2, rmsd 0.8 Å, seq id: 70%; PDB-code: 3WWH from Arthrobacter sp. KNK168[10f], Z-score:
41.8, rmsd 1.8 Å, seq id: 44%).
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A
B
Figure 1. Crystal structure of EX-TA. A: Overview of the EX-TA dimer (chain A in orange,
chain B in turquoise), PLP is indicated in yellow, B: Overview of the EX-TA dimer active site
with the small binding pocket amino acids depicted in orange and the large binding pocket amino
acids in dark grey. The figures were prepared using the program PyMOL.
The active site showed a comparable size to the ones from other fold IV ATAs[10a-b, 10e] and
consisted of a small and a large binding pocket (Figure 1B). In general, the larger pocket
accommodates the bulky substituent of the ketone substrate or the α-amine of the co-substrate.
The small binding pocket harbors the smaller substituent, usually a methyl-group.[4,10] As we
could not obtain structure-ligand complexes by co-crystallization nor by soaking, biarylic
substrates were docked into the structure (Figure 2A). Even though the structure of EX-TA is
similar to other fold IV ATAs (see above), we postulate that the interplay of two structural
features leads to the ability to convert biaryl ketones: (i) reduced charge and, thus, low polarity
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of the active site, and (ii) a methionine at position 47 which probably contributes to position the
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substrate via S-π interactions (Figure 2A).
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Figure 2. Characteristics of EX-TA that enable the conversion of biaryl ketones: (A) M47
helps to position the biaryl ketones (depicted in this Figure is 1a) via S-π interactions; (B) charge
of the EX-TA active site (PDB ID: 6FTE) and (C) active site charge of other fold IV ATAs
(from left to right): Aspergillus terreus AT-TA (PDB ID: 4CE5), Nectria haematococca TA
(PDB ID: 4CMD) and A. fumigatus TA (PDB ID: 4CHI). The figures were prepared using the
program PyMOL.
Biaryl ketones are hydrophobic and uncharged entities and, thus, bind better in an active site
with low polarity. In contrast to other (R)-ATAs, which have an active site with high polarity, the
EX-TA active site shows a reduced overall charge and is therefore better suited to
accommodate the non-polar biaryl moiety of the substrate (compare Figure 2B and C). The
methionine at position 47 could also play a key role to enable the conversion of bulky conjugated
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substrates: The sulfur of this methionine is located within a distance of 4.7 Å from the outer
phenyl-ring and therefore helps to position the biaryl moiety via S-π interactions. To confirm this
hypothesis, this methionine was replaced by a leucine (as found in Aspergillus terreus ATA
(AT-TA) and in A. fumigatus), and indeed, the conversion of 1a into 2a decreased from 83% to
17%.
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Engineering of EX-TA
A consensus design approach was followed to enlarge the small binding pocket of EX-TA for
accommodation of larger substituents than the original methyl group. The small binding pocket
of EX-TA is defined by the amino acids V60, F113, T273, T274 and A275 (Figure 1A).
Multiple sequence alignment with other ATAs showed that F113 is highly conserved, V60 is
rather conserved and also T273, T274 and A275 do not have a lot of variation. If the consecutive
amino acids 273 to 275 are taken together as a motif, just four different combinations exist in
nature (Figure S2). These are TTA, STA, SSA and SSG. TTA is the combination that takes most
space and is the one that is found in EX-TA (and also in the TAs from A. terreus and A.
fumigatus). Hence, the TTA motif was exchanged to STA, SSA and SSG. All variants were
soluble and active. EX-STA emerged with significantly improved activity toward all tested
substrates except 1a (Table 1 and Table S4), while the biaryl ethyl ketone 1d remained
unconverted (Table 1, entry 4). Remarkably, EX-STA preserved the exquisite enantioselectivity
of the wild type enzyme (>99% ee) and the three pyridyl analogues 1h-j exhibited complete
conversion (Table 1, entries 8-10). It seems that this amino acid exchange beneficially impacts
the positioning of the substrates in the large binding pocket.
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The characterization of EX-TA revealed a quite low thermostability in comparison to other
ATAs, especially to AT-TA (Figure S4A). While an incubation at 40°C resulted in a thermal
inactivation half-life t1/2 of 11.3 min for Ex-TA, no decrease of activity could be observed for
AT-TA over the measuring time of 60 min (Figure S4A). To overcome the stability hurdles of
EX-TA, stabilizing interactions between amino acid residues were identified within the
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structure of AT-TA, and consequently introduced into the structure of EX-TA, if they were
absent there. To decrease the number of variants but simultaneously increase the chance of
success, FoldX[29] was used to determine if an amino acid exchange would be likely to increase
or decrease the stability. The best resulting variant EX-5 (for other variants see SI) has five
amino acid exchanges: K317E introduces a new ionic interaction to R67, and new cation-π
interactions were created with the amino acid exchanges K110R, L191F and N249F/E300K
(Figure S3 and Table S5). K110R interacts with Y320, L191F with R86 and R120. N249F and
E300K interact with each other. Indeed, compared to the wild type, the thermal inactivation half-
life t1/2 at 45 °C is improved by 10 min and the thermal inactivation T1/2 after 10 min increased
by 4.4 °C (Figure 3 and Table 2). This thermostable variant EX-5 retained the same excellent
enantioselectivity as the wild type (>99% ee in all cases). Interestingly, however, the substrate
tolerance showed significant differences: for example, EX-5 poorly converted 1a (15%, Table 1,
entry 1) but was very active toward the fluorine-substituted derivative 1e (84% Table 1, entry 5)
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Figure 3. Thermal inactivation properties of wild type EX-TA and variants thereof. (A)
Thermal inactivation half-life t1/2 at 45°C. (B) Thermal inactivation at different temperatures
T1/210. Error bars are the mean SD values of three samples. Solid lines represent the fitted data.
Table 2. Stability constants of wild-type EX-TA and variants thereof.
EX-
wt
EX-
STA
EX-5
EX-
STA5
∆∆Gfold [kcal/mol]
-
-
-0.92
0
-3.03 -4.06
-0.26 -0.21
∆∆Gbinding
[kcal/mol]
T1/210 min [°C]
∆T1/210 min [°C]
t1/245°C [min]
42.5
42.6
0.1
5.9
0.3
46.9
4.4
46.1
3.6
-
5.6
-
15.8
10.2
16.3
10.7
∆t1/245°C [min]
Since EX-STA converted most of the substrates more efficiently than the wild type (TTA) or
EX-SSA and EX-SSG (Table S4), the STA motif was combined with the amino acid exchanges
from the more thermostable variant EX-5. As a result, the combined variant EX-STA5 now
encompasses similar or only slightly lower activity than EX-STA (Table 1) but also shows an
improved stability like EX-5 (Table 2 and Figure 3). These results reveal that the acquisition of
the enhanced enzymatic activity did not have a detrimental effect on stability, which makes the
resulting ATA EX-STA5 a more robust biocatalyst for its implementation in an industrial setting.
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Substrate Scope of EX-TA and its Variants Toward Non-Biaryl Ketones
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8
Once the transamination of biaryl ketones was established employing either EX-TA or its
variants, we sought to deepen the knowledge about the structural requirements of these new
ATAs. Thus, a series of non biaryl ketones was assayed with the EX-wildtype and the three best
variants (Table 3).
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Table 3. Substrate scope of EX-TA and its variants toward non-biaryl ketones.[a]
Conversion (%)
ee
Entry Substrate
(%)[b]
EX-
wt
EX-
STA
EX-
STA5
EX-5
<5
-
O
O
1k
(
)
1
2
3
4
5
6
7
8
<5
-
7
-
<5
-
n.d.[c]
Ph
Ph
1l
(
)
-
O
1m
(
)
15
95
67
-
60
76
66
<5
-
15
55
62
<5
-
60
65
60
-
>99
>99
>99
n.d.[c]
-
Ph
Ph
1n
(
)
O
O
1o
(
)
Ph
O
1p
(
)
O
1q
(
)
-
-
O
1r
(
)
49
94
93
52
>99
[a] Reaction conditions: 20 mM ketone dissolved in DMSO (5% v/v), 100 mM potassium
phosphate buffer, pH 7.5, 1 mM PLP, 0.1 mM NAD+, 20 U ATA, 130 mM D-alanine, 60 mM
glucose, 30 U GDH and 90 U LDH, shaken for 24 h at 250 rpm and 30 ºC. Conversions were
measured by HPLC. [b] Measured by chiral HPLC; the ee is the same for all four enzymes and
the (R)-configuration was obtained in all cases. [c] Not detected due to low activity.
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As expected, conversions of propiophenone (1k) stayed rather low and none of the ATAs was
capable to convert 1-phenylbutan-2-one (1l) which confirms the size restrictions of the small
binding pocket pointed out before (Table 3, entries 1-2). In contrast, different methyl monoaryl
ketones such as acetophenone (1m), 1-phenylpropan-2-one (1n) and 4-phenylbutan-2-one (1o)
with variable length of the alkyl chain exhibited moderate to good conversions depending on the
ATA variant and ketone considered (Table 3, entries 3-6). Further extension to benzo-fused
cyclic ketones revealed that those with the carbonyl moiety contiguous to the methylene bridge
were unreactive (1p-q, Table 3, entries 6-7). However, 2-tetralone (1r) afforded excellent
conversions catalyzed by both EX-STA and EX-STA5 (Table 3, entry 8).
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Preparative-scale Biaryl Amine Synthesis
Taking the EX-STA-catalyzed transamination of 1i as a model reaction (Table 1, entry 9),
several process parameters were investigated on an analytical scale. In detail, the formulation of
the biocatalyst, the amino donor, the concentration of substrate and the enzyme loading were
assessed. Importantly, the variants EX-5, EX-STA and EX-STA5 retained their activity as a
lyophilized powder of the lysate in contrast to the wild type enzyme (Figure S5). In addition, the
variants also accepted isopropylamine as an amino donor (Figure S6) although with lower
conversions than those measured with alanine. By using EX-STA as a lyophilized powder the
upper limit for the substrate concentration was established at 75 mM (Figure S7). Finally, the
enzyme loading was optimized to 3.2 U/mg of substrate (Figure S8).
To illustrate the synthetic applicability of the new ATAs, a preparative-scale biotransformation
was carried out. The target biotransformation was accomplished at 100 mg-scale and 75 mM of
1i to afford enantiopure (R)-2i in excellent isolated yield (96%) after extractive work up and
without need for further purification (Scheme 2).
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O
NH2
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EX-TA/ PLP
D-Ala, LDH/GDH system
KPi buffer pH 7.5, DMSO
30 ºC, 250 rpm
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1i
2i
N
N
75 mM
(100 mg)
96% yield
>99% ee
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Scheme 2. Preparative-scale transamination of 1i catalyzed by EX-TA.
CONCLUSION
In summary, we report a new (R)-selective ATA which naturally converts biaryl ketones to the
corresponding enantiomerically pure amines. With the help of its crystal structure, structural
features were identified which could explain its unique ability. The activity and stability of this
new ATA was further improved by protein engineering, resulting in improved biocatalysts
whose applicability was illustrated through a preparative-scale example. Our results highlight the
value of nature’s biodiversity and extreme environments as an ever-increasing source of new
genomes and metagenomes to identify unprecedented catalytic activities which can lead to novel
industrially robust biocatalysts.
ASSOCIATED CONTENT
Supporting Information.
Supporting Tables: conserved motifs of fold IV PLP-dependent enzymes, list of primers, protein
crystallography data;
Additional experimental procedures and data for protein engineering;
Supporting Figures: omit electron density of PLP in the structure, biocatalyst formulation study,
transamination of 1i using isopropylamine as amino donor, substrate loading studies, enzyme
loading studies;
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Detailed results of enzymatic transamination of ketones 1a-j; assignments of the absolute
configurations, spectroscopic data of amines 2a-b, e-j; HPLC analytical data; copies of chiral-
HPLC chromatograms; copies of NMR spectra
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This information is available free of charge on ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
* Email: kerstin.steiner11@googlemail.com
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
The Biocascades project is supported by the European Commission under the Horizon 2020
program through the Marie Skłodowska-Curie actions: ITN-EID under the Environment Cluster
(Grant Number 634200).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The Biocascades project is supported by the European Commission under the Horizon 2020
program through the Marie Skłodowska-Curie actions: ITN-EID under the Environment Cluster
(Grant Number 634200). We thank Math Boesten for assistance with HPLC analysis.
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REFERENCES
5
6
7
8
(1) Jarvis, L. M. The year in new drugs. Chem. Eng. News 2016, 94, 12–17.
(2) Kelly, S. A.; Pohle, S.; Wharry, S.; Mix, S.; Allen, C. C. R.; Moody, T. S.; Gilmore, B. F.
Application of ω‐Transaminases in the Pharmaceutical Industry. Chem. Rev. 2018, 118, 349-
367.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3) Malik, M. S.; Park, E. S.; Shin, J. S. Features and technical applications of ω-transaminases.
Appl. Microbiol. Biotechnol. 2012, 94, 1163–1171.
(4) Höhne, M.; Schätzle, S.; Jochens, H.; Robins, K.; Bornscheuer, U. T. Rational assignment of
key motifs for function guides in silico enzyme identification. Nat. Chem. Biol. 2010, 6, 807–
813.
(5) (a) Dourado, D. F. A. R.; Pohle, S.; Carvalho, A. T. P.; Dheeman, D. S.; Caswell, J. M.;
Skvortsov, T.; Miskelly, I.; Brown, R. T.; Quinn, D. J.; Allen, C. C. R.; Kulakov, L.; Huang,
M.; Moody, T. S. Rational Design of a (S)-Selective-Transaminase for Asymmetric Synthesis
of (1S)-1-(1,1′-biphenyl-2-yl)ethanamine. ACS Catal. 2016, 6, 7749–7759; (b) Pavlidis, I. V.;
Weiß, M. S.; Genz, M.; Spurr, P.; Hanlon, S. P.; Wirz, B.; Iding, H.; Bornscheuer, U. T.
Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral
amines. Nat. Chem. 2016, 8, 1–7; (c) Han, S. W.; Park, E. S.; Dong, J. Y.; Shin, J. S.
Mechanism-Guided Engineering of ω-Transaminase to Accelerate Reductive Amination of
Ketones. Adv. Synth. Catal. 2015, 357,1732–1740; (d) Weiß, M. S.; Pavlidis, I. V.; Spurr, P.;
Hanlon, S. P.; Wirz, B.; Iding, H.; Bornscheuer, U. T. Amine transaminase engineering for
spatially bulky substrate acceptance. ChemBioChem 2017, 18, 1022–1026; (e) Weiß, M. S.;
Pavlidis, I. V.; Spurr, P.; Hanlon, S. P.; Wirz, B.; Iding, H.; Bornscheuer, U. T. Protein-
engineering of an amine transaminase for the stereoselective synthesis of a pharmaceutically
ACS Paragon Plus Environment
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Page 27 of 36
ACS Catalysis
1
2
3
4
5
6
7
8
9
relevant bicyclic amine. Org. Biomol. Chem. 2016, 14, 10249–10254; (f) Nobili, A.; Steffen-
Munsberg, F.; Kohls, H.; Trentin, I.; Schulzke, C.; Höhne, M.; Bornscheuer, U. T.
Engineering the Active Site of the Amine Transaminase from Vibrio fluvialis for the
Asymmetric Synthesis of Aryl– Alkyl Amines and Amino Alcohols. ChemCatChem 2015, 7,
757–760.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(6) Calvelage, S.; Dörr, M.; Höhne, M.; Bornscheuer, U. T. A Systematic Analysis of the
Substrate Scope of (S)- and (R)-selective Amine Transaminases. Adv. Synth. Catal. 2017,
359, 4235-4243.
(7) Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J.
C.; Krebber, A.; Fleitz, F. J.; Brands, J.; Devine, P. N.; Huisman, G. W.; Hughes, G. J.
Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin
Manufacture. Science 2010, 329, 305–309.
(8) (a) Horton, D. A.; Bourne, G. T.; Smythe, M. L. The Combinatorial Synthesis of Bicyclic
Privileged Structures or Privileged Substructures. Chem. Rev. 2003, 103, 893–930; (b) Yet,
L. Biaryls in Privileged Structures in Drug Discovery: Medicinal Chemistry and Synthesis,
John Wiley & Son, Hoboken, NJ, USA, 2018. P. 83-154
(9) Dawood, A. W. D.; Bassut, J.; de Souza, R. O. M. A.; Bornscheuer, U. T. Combination of the
Suzuki–Miyaura Cross‐Coupling Reaction with Engineered Transaminases. Chem. Eur. J.
2018, 24, 16009-16013.
(10) (a) Sayer, C.; Martinez-Torres, R. J.; Richter, N.; Isupov, M. N.; Hailes, H. C.;
Littlechild, J. A.; Ward, J. M. The substrate specificity, enantioselectivity and structure of the
(R)-selective amine:pyruvate transaminase from Nectria haematococca. FEBS J. 2014, 281,
2240–2253; (b) Thomsen, M.; Skalden, L.; Palm, G. J.; Höhne, M.; Bornscheuer, U. T.;
ACS Paragon Plus Environment
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ACS Catalysis
Page 28 of 36
1
2
3
4
5
6
7
8
9
Hinrichs, W. Crystallographic characterization of the (R)-selective amine transaminase from
Aspergillus fumigatus. Acta Crystallogr. Sect. D Biol. Crystallogr. 2014, 70, 1086–1093; (c)
Schätzle, S.; Steffen-Munsberg, F.; Thontowi, A.; Höhne, M.; Robins, K.; Bornscheuer, U.
T. Enzymatic Asymmetric Synthesis of Enantiomerically Pure Aliphatic, Aromatic and
Arylaliphatic Amines with (R)-Selective Amine Transaminases. Adv. Synth. Catal. 2011,
353, 2439–2445; (d) Jiang, J.; Chen, X.; Zhang, D.; Wu, Q.; Zhu, D. Characterization of (R)-
selective amine transaminases identified by in silico motif sequence blast. Appl. Microbiol.
Biotechnol. 2015, 99, 2613–2621; (e) Łyskowski, A.; Gruber, C.; Steinkellner, G.;
Schürmann, M.; Schwab, H.; Gruber, K.; Steiner, K. Crystal Structure of an (R)-Selective ω-
Transaminase from Aspergillus terreus. PLoS One 2014, 9, e87350. (f) Guan, L. J.; Ohtsuka,
J.; Okai, M.; Miyakawa, T.; Mase, T.; Zhi, Y.; Hou, F.; Ito, N.; Iwasaki, A.; Yasohara,
Y.; Tanokura, M. A new target region for changing the substrate specificity of amine
transaminases. Sci. Rep. 2015, 5, 10753.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(11) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment
search tool. J. Mol. Biol. 1990, 215, 403-410.
(12) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular
Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 2013, 30, 2725–9.
(13) De Hoog, G. S.; Zeng, J. S.; Harrak, M. J.; Sutton, D. A. Exophiala xenobiotica sp. nov.,
an opportunistic black yeast inhabiting environments rich in hydrocarbons. Antonie van
Leeuwenhoek, Int. J. Gen. Mol. Microbiol. 2006, 90, 257–268.
(14) Balzer, D.; Ziegelin, G.; Pansegrau, W.; Kruft, V.; Lanka, E. KorB protein of
promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for
conjugative plasmid transfer. Nucleic Acids Res. 1992, 20, 1851–8.
ACS Paragon Plus Environment
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Page 29 of 36
ACS Catalysis
1
2
3
4
5
6
7
8
9
(15) Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M. R.; Appel, R. D.;
Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server. Proteomics
Protoc. Handb. 2005, 571–607.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(16) Kabsch, W. XDS.Acta Crystallogr. D. Biol. Crystallogr. 2010, 66, 125–32.
(17) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.;
Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658–674.
(18) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot.
Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66, 486–501.
(19) Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R.
A.; Winn, F. Long, M. D.; Vagin, A. A. REFMAC5 for the refinement of macromolecular
crystal structures. Acta Crystallogr. Sect. D Biol. Crystallogr. 2011, 67, 355–367.
(20) (a) Chen, V. B.; Arendall, W. B.; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral,
G. J.; Murray, L. W; Richardson, J. S.; Richardson, D. C. MolProbity: all-atom structure
validation for macromolecular crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr.
2010, 66, 12–21. (b) Williams, C. J.; Headd, J. J.; Moriarty, N. W.; Prisant, M. G.; Videau, L.
L.; Deis, L. N.; Verma, V.; Keedy, D. A.; Hintze, B. J.; Chen, V. B.; Jain S.; Lewis, S. M.;
Arendall, W. B.; Snoeyink, J.; Adams, P. D.; Lovell, S. C.; Richardson, J. S.; Richardson, D.
C. MolProbity: More and better reference data for improved all-atom structure validation.
Protein Sci. 2017, 27, 293-315.
(21) Krissinel, E.; Henrick K. Inference of Macromolecular Assemblies from Crystalline
State. J. Mol. Biol. 2007, 372, 774–79.
ACS Paragon Plus Environment
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