Engineering the active site of the amine transaminase from vibrio fluvialis for the asymmetric synthesis of aryl-alkyl amines and amino alcohols

Article abstract of DOI:10.1002/cctc.201403010

Although the amine transaminase from Vibrio fluvialis has often been applied as a catalyst for the biocatalytic preparation of various chiral primary amines, it is not suitable for the transamination of α-hydroxy ketones and aryl-alkyl ketones bearing an alkyl substituent larger than a methyl group. We addressed this problem through a systematic mutagenesis study of active site residues to expand its substrate scope towards two bulky ketones. We identified two mutants (F85L/V153A and Y150F/V153A) showing 30-fold increased activity in the conversion of (S)-phenylbutylamine and (R)-phenylglycinol, respectively. Notably, they facilitated asymmetric synthesis of these amines with excellent enantiomeric purities of 98 ee. Excavating the active site: The constrained active site of the amine-transaminase from Vibrio fluvialis was engineered in order to achieve the conversion of bulky ketones. This led to the discovery of mutants with a 30-fold increase in activity which enabled the asymmetric synthesis of (S)-phenylbutylamine and (R)-phenylglycinol

Full text of DOI:10.1002/cctc.201403010

DOI: 10.1002/cctc.201403010
Communications
Engineering the Active Site of the Amine Transaminase
from Vibrio fluvialis for the Asymmetric Synthesis of Aryl–
Alkyl Amines and Amino Alcohols
Alberto Nobili,[a] Fabian Steffen-Munsberg,[a, b] Hannes Kohls,[a] Ivan Trentin,[a]
Carola Schulzke,[a] Matthias Hçhne,[a] and Uwe T. Bornscheuer*[a]
Although the amine transaminase from Vibrio fluvialis has
often been applied as a catalyst for the biocatalytic prepara-
tion of various chiral primary amines, it is not suitable for the
transamination of a-hydroxy ketones and aryl-alkyl ketones
bearing an alkyl substituent larger than a methyl group. We
addressed this problem through a systematic mutagenesis
study of active site residues to expand its substrate scope to-
wards two bulky ketones. We identified two mutants (F85L/
V153A and Y150F/V153A) showing 30-fold increased activity in
the conversion of (S)-phenylbutylamine and (R)-phenylglycinol,
respectively. Notably, they facilitated asymmetric synthesis of
these amines with excellent enantiomeric purities of 98%ee.
despite the progress of first engineering studies.[4] The crystal
structures of several (S)-selective ATAs were solved recently, en-
abling a detailed understanding of the mechanism of substrate
binding.[5]
Both (R)- and (S)-selective ATAs that were found in nature
possess a large and a small pocket in their active sites (Fig-
ure 1a).[5a,6] Although the large pocket can accommodate sub-
stituents with a rather broad size distribution, such as small
alkyl to naphthyl groups, the small pocket creates a strict steric
constraint: if the size of the small substituent exceeds that of
a methyl group, activity drops significantly.[6] For instance, ke-
tones with a hydroxymethyl group as small substituent are
hardly accepted.[7] This active site architecture limits the sub-
strate scope, but at the same time contributes to the usually
high enantioselectivity of these ATAs.
Enantiomerically pure amines and amino alcohols play a funda-
mental role in the pharmaceutical industry. One in four of the
200 top-sold drugs contains a chiral amine moiety and these
drugs had a total market value of more than 88 billion USD in
2013 according to Weber and Sedelmeier.[1] When it comes to
the choice of the synthetic strategy for the preparation of the
amine building blocks, amine transaminases (ATAs) are increas-
ingly recognized as an attractive option as they facilitate
a one-step asymmetric synthesis starting from the correspond-
ing prochiral ketone.[2] A very impressive example is the appli-
cation of an engineered (R)-selective ATA from Arthrobacter sp.
(ATA117-mut), which is currently being used for the production
of sitagliptin, the active ingredient of the drugs Januvia and Ja-
numet.[3] This example demonstrates the importance of protein
engineering of wild-type amine transaminases to expand their
limited substrate scope. Known wild-type ATAs are not able to
convert bulky compounds demanded by the pharmaceutical
industry. Compared to the success story of engineered (R)-se-
lective transaminases with relaxed substrate specificity, (S)-se-
lective ATAs that convert a range of bulky ketones with similar
efficiency as the engineered ATA117-mut are still not available,
Midelfort et al.[4a] and Park et al.[4b] recently reported the first
attempts of rational engineering: they identified key residues
via bioinformatic methods or structural inspection and investi-
gated up to two substitutions per position by site-directed
mutagenesis to achieve the transamination of their bulky
target ketones. By combining eight mutations in Vibrio fluvialis
ATA, a b-keto ester bearing a long (6 carbon) alkyl chain could
be converted employing 1-phenylethylamine 1b as amino
donor, affording the amine imagabalin at 28% yield via asym-
metric synthesis. A single mutant in Paracoccus denitrificans
ATA[4b] showed increased activity in the deamination of 1-alkyl
substituted benzyl amines and the amination of 2-oxo-octa-
noate. Interestingly, this study showed that larger n-alkyl sub-
stituents are accepted in the small binding pocket if the sub-
strate bears an a-carboxylate functional group instead of
a large hydrophobic substituent such as a phenyl group.
Despite these first successes, further efforts are needed to
create an (S)-selective ATA that is useful for asymmetric synthe-
sis of bulky amines. In the present study, we systematically ad-
dress this problem by a (partial) saturation mutagenesis of all
amino acids that form the small binding pocket of the ATA of
Vibrio fluvialis.
[a] A. Nobili, F. Steffen-Munsberg, H. Kohls, I. Trentin, Prof. Dr. C. Schulzke,
Prof. Dr. M. Hçhne, Prof. Dr. U. T. Bornscheuer
Institute of Biochemistry, University of Greifswald
Felix-Hausdorff Str. 4, 17487 Greifswald (Germany)
Fax: (+49)3834-86-794367
We employed 1-phenylbutane-1-one 2a and the hydroxy
ketone 2-hydroxyacetophenone 3a as model substrates
(Table 1). The amine product (R)-phenylglycinol 3b is a building
block for many important pharmaceuticals, such as an inhibitor
of the 3-phosphoinositide-dependent protein kinase-1 (PDK1),
which was identified as a target enzyme for cancer therapy.[8]
Additionally, 3b is applied as a chiral auxiliary in the synthesis
of some of the top selling drugs, saxagliptin[9] (treatment of
type 2 diabetes), femoxetine and paroxetine[10] (antidepres-
E-mail: uwe.bornscheuer@uni-greifswald.de
[b] F. Steffen-Munsberg
KTH Royal Institute of Technology, School of Biotechnology
Division of Industrial Biotechnology
AlbaNova University Center, SE-106 91 Stockholm (Sweden)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201403010.
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3b would also lead to higher activity in the deamination reac-
tion, and thus be detected during screening.
When pyruvate is used as the amino acceptor, the reaction
is virtually irreversible, reducing the screening time and reac-
tion complexity compared to asymmetric synthesis. As this
assay works with both enantiomers, we were also able to de-
termine if a mutation affected the enzyme’s enantioselectivity.
Based on the crystal structure of the transaminase of Vibrio flu-
vialis (PDB-code: 4E3Q), the small binding pocket, located at
the interface of the homodimer, is composed of eight residues
that completely surround the cofactor, from Tyr150 at the
active site entrance (re-face of PLP[12]) to the catalytic Lys285
on the other (si-) side (Figure 1B, the residues that belong to
the second monomer are indicated with an asterisk).[4a] Resi-
dues Tyr165, Tyr150, Phe19, Phe85*, and Phe86* create a con-
tinuous p–p stacked shell and thus form a relatively hydropho-
bic environment. Three of the eight residues, Gly320*,
Phe321*, and Thr322*, were excluded in our mutagenesis
strategy, as they are known as the “phosphate-binding cup”[13]
and are responsible for the coordination of the PLP phosphate
group. All the other positions (Phe19, Leu56, Phe85*, Tyr150
and Val153) were sifted through for improved properties in
the transamination between the enantiopure amines and pyru-
vate. In a first step, we generated libraries with a (partial) satu-
ration at each position. The allowed residues in the partially sa-
turated libraries included those amino acids that aim to create
more space in the pocket, while still maintaining structural sol-
idity (Figure 1B). The quality of each library was checked to
ensure 99% library coverage during the screening,[14] then
each library was screened using the modified acetophenone
assay.[11] Variants with improved activities were purified and fur-
ther investigated to confirm their improved properties. Finally,
the best mutants were combined to elucidate positive additive
effects. When positions 19 and 56 were randomized, no im-
proved variants were found for the substrates tested (data not
shown).
Figure 1. Active site architecture of Vibrio fluvialis ATA (PDB-code: 4E3Q).
A) Schematic drawing of residues that form the large and small binding
pocket around the external aldimine intermediate (PLP-Schiff’ base with (S)-
1-phenylethylamine). B) View of the small binding pocket. The side-chains of
five residues form a hydrophobic shell by p-p stacking interactions. Y165
and F86* are omitted for clarity. The catalytic lysine and PMP are shown as
gray sticks; the targeted residues for mutagenesis and their surface are
shown in orange and are labeled by one-letter abbreviation and number.
The libraries were constructed by choosing those residues that were poten-
tially able to decrease the steric hindrance in the pocket, that is, smaller and
aliphatic residues. Residues with different chemical properties derive from
the selection of the randomized codons (see the Supporting Information).
The phosphate-binding cup is shown in blue.
Single mutations at positions 85, 150, and 153 led to up to
40-fold improvements. For the conversion of 3b, the results
suggest that the positioning of its hydroxyl group in the small
binding pocket is hindered by the presence of the Tyr150 in
the wild-type scaffold (Figure 1), whose hydroxyl group occu-
pies the required space. The simple substitution Y150F im-
proves the template’s performance 23-fold, whereas the best
mutant discovered in this project is the double mutant
Y150M/V153A for a final improvement of the reaction velocity
of 53-fold. By combining the mutations F85L and V153A it was
possible to achieve a 26-fold improvement towards 2b. We
then tested the ability of the best mutants to catalyze the
asymmetric synthesis of our target amines, using the LDH/GDH
system to shift the equilibrium.[15]
sants). These substrates exert an increased steric demand on
the small binding pocket compared to the well-known aceto-
phenone 1a, which is often employed as a benchmark sub-
strate. Furthermore, (S)-phenylbutylamine 2b and (R)-phenyl-
glycinol 3b facilitate an easy activity screening owing to the
increase in UV-absorption upon deamination to their corre-
sponding ketones 2a and 3a. Hence, the acetophenone
assay[11] could be used with slight modifications to screen for
active variants with high throughput and sensitivity (see Fig-
ure S1 in the Supporting Information). We assumed that muta-
tions which would facilitate asymmetric synthesis of 2b and
Regrettably, the mutants carrying the mutation Y150M had
a decreased activity towards 2a. The Y150M-containing mu-
tants were not among the best mutants for the transamination
of 3a, either. Consequently, we investigated with increased
enzyme concentrations all the different improved variants
identified by the screening in the asymmetric synthesis reac-
tion and we found that variant Y150F/V153A formed 3b with
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was increased by up to 6-fold in
the synthesis of l-Leu, l-Ile, and
l-Val (Table S2).
Table 1. Specific activities of the purified wild-type and variants identified during the screening.
Our results demonstrate the
difficulty of broadening the sub-
strate specificity of (S)-selective
ATAs. All amino acids that con-
tribute to the small binding
pocket are located at the dimer
interface of the enzymes (except
Y150) and they are placed on
different loops. Although the
placement of smaller residues in
positions 19, 56, and 85 would
theoretically excavate the small
binding pocket and provide
more space for bulky substrates,
mutations at these sites seem to
affect other properties of the
enzyme (such as flexibility) im-
portant for folding, catalysis or
stability. Other than the effect of
F85L on 2b, all mutations in
these positions were detrimen-
tal. This supports previous find-
ings by Park et al. and Humble
et al. where mutations at the
structurally equivalent positions
1b
2b
3b
[a]
[a]
[a]
Variant
SA [UmgÀ1
]
SA [UmgÀ1
]
Conv [%][b]
SA [UmgÀ1
]
Conv [%][b]
VF-wt
F85L
Y150F
Y150M
1.33Æ0.02
0.58Æ0.01
0.97Æ0.02
0.68Æ0.03
5.59Æ0.07
0.55Æ0.02
0.69Æ0.04
2.38Æ0.14
1.60Æ0.02
1.36Æ0.03
0.98Æ0.01
0.47Æ0.01
0.19Æ0.00
0.32Æ0.00
0.22Æ0.01
0.39Æ0.01
3.36Æ0.03
0.35Æ0.00
0.74Æ0.01
4.99Æ0.07
0.82Æ0.02
0.65Æ0.02
1.55Æ0.03
0.02Æ0.00
19
51
20
8
71
52
14
93
44
8
0.01Æ0.00
0.00Æ0.00
0.23Æ0.00
0.41Æ0.02
0.11Æ0.00
0.07Æ0.00
0.35Æ0.00
0.03Æ0.01
0.34Æ0.01
0.53Æ0.03
0.14Æ0.01
0.00Æ0.00
14
0
93
47
49
30
0
V153A
F85L/Y150F
F85L/Y150M
F85L/V153A
Y150F/V153A
Y150M/V153A
F85L/Y150F/V153A
F85L/Y150M/V153A
0
92
23
32
0
79
1
[a] SA= Specific activity: The reaction was followed at 245, 242, and 252 nm for the detection of 1a, 2a, and
3a, respectively, using 2.5 mm of the amino donor and 2.5 mm of pyruvate in 50 mm HEPES buffer pH 7.5 con-
taining 1.66% DMSO at 308C. The activities were calculated as UmgÀ1 (purified enzyme). One Unit is defined
as the conversion of 1 mmol of product per minute. Values and standard deviations given are based on three
measurements. [b] Conversion reached in asymmetric synthesis: Reaction conditions for the synthesis of 2b:
0.5 mgmLÀ1 purified enzyme, 1 mL reaction volume, 10 mm 2a, 150 mmL-alanine, 30% DMSO, 14 days, 308C,
50 mm HEPES buffer pH 7.5, 0.1 mm PLP. Reaction conditions for the synthesis of 3b: 0.5 mgmLÀ1 purified
enzyme, 1 mL reaction volume, 10 mm 3a, 250 mmL-alanine, 10% DMSO, 4 days, 308C, 50 mm HEPES buffer
pH 7.5, 0.1 mm PLP. Both reactions were followed via GC analysis using a Hydrodex-b-TBDAC chiral column and
in both cases the LDH/GDH system was used to shift the equilibrium.[15]
88% conversion after 24 h. In agreement with the previous ob-
servations, the asymmetric synthesis of 3b is possible when
the active site is freed from the Tyr150’s hydroxyl group and
Y150F is now the key mutation to accomplish this transamina-
tion. Variant F85L/V153A afforded 2b with quantitative conver-
sion after 5 days (Table S1).
led to a drastic drop in activity in the ATAs from Paracoccus de-
nitrificans and Chromobacterium violaceum.[4b,16]
Most of the variants screened from those libraries showed
little-to-no residual activity in our initial screening assays com-
pared to the wild-type. In position 150, a Phe or Tyr is found in
virtually all ATA-sequences, as this position is important for the
positioning and p-stacking with PLP. The presence of the Tyr
hydroxyl group, however, determines the acceptance of the
hydroxylated substrates 3a and 3b in the forward or backward
reaction, respectively. This result is in agreement with those
presented in an independent study performed on the ATA
from Chromobacterium violaceum, and published by
Deszcz et al. contemporaneously to ours.[17]
The asymmetric synthesis of 2b and 3b was then confirmed
on a semi-preparative scale (Table 2) employing 0.2 mmol
ketone and 0.2 mol% catalyst. We also observed a beneficial
effect of the above-identified mutations on the activity of the
ATA towards branched-chain a-keto acids, where the activity
The different activities of Y150F- and Y150M-con-
Table 2. Asymmetric synthesis results with the best (purified) variants.
taining variants in the kinetic resolution or asymmet-
ric synthesis underline the importance of screening
Mutant
Product
Conv.
[%]
ee
[%]
Yield
[%]
variants under conditions as close to the desired syn-
thetic application as possible. V153A showed the
largest contribution towards a relaxed active site able
to accept bulkier substrates, which was also observed
by Park and coworkers.[4b] This second shell residue is
not in contact with the bound substrate, but its mu-
tation to alanine might increase the active site’s flexi-
bility.
F85L/V153A
2b
3b
>98[a]
>98[b]
98
98
53
Y150F/V153A
60
The reaction progress was followed via TLC analysis (0.2 mm detection limit of the
ketone). To shift the equilibrium, the LDH/GDH system was applied.[15] [a] Reaction
conditions: 1 mgmLÀ1 purified enzyme, 20 mL reaction volume, 10 mm 2a, 150 mmL-
alanine, 30% DMSO, 14 days, 308C, 50 mm HEPES buffer pH 7.5, 0.1 mm PLP. [b] Reac-
tion conditions: 1 mgmLÀ1 purified enzyme 20 mL reaction volume, 10 mm 3a,
250 mmL-alanine, 10% DMSO, 3 days, 308C, 50 mm HEPES buffer pH 7.5, 0.1 mm PLP.
Finally, we conclude that 1) there is no easy solu-
tion to generally expand the substrate scope of
Vibrio fluvialis ATA by modifying single residues of
the small binding pocket, and 2) creating space is
not sufficient to yield an efficient amine transaminase
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[4] a) K. S. Midelfort, R. Kumar, S. Han, M. J. Karmilowicz, K. McConnell, D. K.
Gehlhaar, A. Mistry, J. S. Chang, M. Anderson, A. Villalobos, J. Minshull,
S. Govindarajan, J. W. Wong, Protein Eng. Des. Sel. 2013, 26, 25–33;
b) E.-S. Park, S.-R. Park, S.-W. Han, J.-Y. Dong, J.-S. Shin, Adv. Synth. Catal.
2014, 356, 212–220.
activity. Instead, second shell residues should be included in
further mutagenesis studies. The prediction of further useful
mutations is not possible at the moment, because the underly-
ing factors governing catalytic efficiency are not yet under-
stood and hence, depending on the chemical structure of the
substrate, different mutations are needed to enhance activity.
[5] a) F. Steffen-Munsberg, C. Vickers, A. Thontowi, S. Schꢁtzle, T. Meinhardt,
M. S. Humble, H. Land, P. Berglund, U. T. Bornscheuer, M. Hçhne, Chem-
CatChem 2013, 5, 154–157; b) F. Steffen-Munsberg, C. Vickers, A. Thon-
towi, S. Schꢁtzle, T. Tumlirsch, M. S. Humble, H. Land, P. Berglund, U. T.
Bornscheuer, M. Hçhne, ChemCatChem 2013, 5, 150–153.
[6] J.-S. Shin, B.-G. Kim, J. Org. Chem. 2002, 67, 2848–2853.
[7] F. G. Mutti, W. Kroutil, Adv. Synth. Catal. 2012, 354, 3409–3413.
[8] D. A. Erlanson, J. W. Arndt, M. T. Cancilla, K. Cao, R. A. Elling, N. English,
J. Friedman, S. K. Hansen, C. Hession, I. Joseph, G. Kumaravel, W.-C. Lee,
K. E. Lind, R. S. McDowell, K. Miatkowski, C. Nguyen, T. B. Nguyen, S.
Park, N. Pathan, D. M. Penny, M. J. Romanowski, D. Scott, L. Silvian, R. L.
Simmons, B. T. Tangonan, W. Yang, L. Sun, Bioorg. Med. Chem. Lett. 2011,
21, 3078–3083.
Experimental Section
All experimental details are presented in the Supporting
Information.
Acknowledgements
FS thanks the “Fonds der Chemischen Industrie” for financial sup-
port. We especially thank the European Union (KBBE-2011-5,
grant No. 289350) for financial support within the European
Union Seventh Framework Programme. We also thank Dr. Ioan-
nis V. Pavlidis and Anders M. Knight for their useful feedback
during the preparation of the manuscript.
[9] D. J. Augeri, J. A. Robl, D. A. Betebenner, D. R. Magnin, A. Khanna, J. G.
Robertson, A. Wang, L. M. Simpkins, P. Taunk, Q. Huang, S. P. Han, B.
Abboa-Offei, M. Cap, L. Xin, L. Tao, E. Tozzo, G. E. Welzel, D. M. Egan, J.
Marcinkeviciene, S. Y. Chang, S. A. Biller, M. S. Kirby, R. A. Parker, L. G.
Hamann, J. Med. Chem. 2005, 48, 5025–5037.
[10] M. Amat, J. Bosch, J. Hidalgo, M. Canto, M. Perez, N. Llor, E. Molins, C.
Miravitlles, M. Orozco, J. Luque, J. Org. Chem. 2000, 65, 3074–3084.
[11] S. Schꢁtzle, M. Hçhne, E. Redestad, K. Robins, U. T. Bornscheuer, Anal.
Chem. 2009, 81, 8244–8248.
[12] K. Soda, T. Yoshimura, N. Esaki, Chem. Rec. 2001, 1, 373–384.
[13] M. S. Humble, K. E. Cassimjee, M. Hakansson, Y. R. Kimbung, B. Walse, V.
Abedi, H. J. Federsel, P. Berglund, D. T. Logan, FEBS J. 2012, 279, 779–
792.
[14] S. Kille, C. G. Acevedo-Rocha, L. P. Parra, Z.-G. Zhang, D. J. Opperman,
M. T. Reetz, J. P. Acevedo, ACS Synth. Biol. 2013, 2, 83–92.
[15] D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell, W. Kroutil, Adv. Synth.
Catal. 2008, 350, 2761–2766.
[16] M. S. Humble, K. E. Cassimjee, V. Abedi, H.-J. Federsel, P. Berglund,
ChemCatChem 2012, 4, 1167–1172.
[17] D. Deszcz, P. Affaticati, A. Gegel, J. M. Ward, H. C. Hailes, P. A. Dalby,
FEBS J. in press.
Keywords: amine transaminase
engineering · substrate scope
·
biocatalysis
·
protein
[1] F. Weber, G. Sedelmeier, Nachr. Chem. 2013, 61, 528–529.
[2] a) H. Kohls, F. Steffen-Munsberg, M. Hçhne, Curr. Opin. Chem. Biol. 2014,
19, 180–192; b) W. Kroutil, E.-M. Fischereder, C. S. Fuchs, H. Lechner,
F. G. Mutti, D. Pressnitz, A. Rajagopalan, J. H. Sattler, R. C. Simon, E. Siiro-
la, Org. Process Res. Dev. 2013, 17, 751–759; c) M. Hçhne, U. T. Born-
scheuer, in Enzymes in Organic Synthesis (Eds.: O. May, H. Grçger, W.
Drauz), Wiley-VCH, Weinheim, 2012, pp. 779–820; d) D. Ghislieri, N.
Turner, Top. Catal. 2014, 57, 284–300; e) D. Koszelewski, K. Tauber, K.
Faber, W. Kroutil, Trends Biotechnol. 2010, 28, 324–332.
[3] C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R. Jarvis,
J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P. N. Devine, G. W. Huis-
man, G. J. Hughes, Science 2010, 329, 305–309.
Received: December 11, 2014
Published online on && &&, 0000
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COMMUNICATIONS
Excavating the active site: The con-
strained active site of the amine-trans-
aminase from Vibrio fluvialis was engi-
neered in order to achieve the conver-
sion of bulky ketones. This led to the
discovery of mutants with a 30-fold in-
crease in activity which enabled the
asymmetric synthesis of (S)-phenylbutyl-
amine and (R)-phenylglycinol
A. Nobili, F. Steffen-Munsberg, H. Kohls,
I. Trentin, C. Schulzke, M. Hçhne,
U. T. Bornscheuer*
&& – &&
Engineering the Active Site of the
Amine Transaminase from Vibrio
fluvialis for the Asymmetric Synthesis
of Aryl–Alkyl Amines and Amino
Alcohols
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