Stereoelectronic effects in the reaction of aromatic substrates catalysed by: Halomonas elongata transaminase and its mutants

Article abstract of DOI:10.1039/c6ob01629d

A transaminase from Halomonas elongata and four mutants generated by an in silico-based design were recombinantly produced in E. coli, purified and applied to the amination of mono-substituted aromatic carbonyl-derivatives. While benzaldehyde derivatives were excellent substrates, only NO₂-acetophenones were transformed into the (S)-amine with a high enantioselectivity. The different behaviour of wild-type and mutated transaminases was assessed by in silico substrate binding mode studies.

Full text of DOI:10.1039/c6ob01629d

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Stereoelectronic effects in the reaction of aromatic substrates
catalysed by Halomonas elongata transaminase and its mutants
Received 00th January 20xx,
Accepted 00th January 20xx
‡a,b
‡a
b
a,c
Martina Letizia Contente , Matteo Planchestainer , Francesco Molinari *, Francesca Paradisi *
DOI: 10.1039/x0xx00000x
www.rsc.org/
A transaminase from Halomonas elongata and four mutants electrophilic and the reaction is slow and thermodynamically
8
generated by an in silico-based design, were recombinantly unfavoured. This aspect heavily influences the wide applicability of
9
produced in E. coli, purified and applied to the amination of these enzymes as an alternative to conventional synthesis. On the
mono-substituted
benzaldehyde derivatives resulted excellent substrates, only NO
acetophenones were transformed into the (S)-amine with high gauged after investigation of the enzyme-substrate binding mode.
aromatic
carbonyl-derivatives.
While other hand, unfavourable steric interactions between the enzyme
2
-
and the substrate may hamper the reactivity and can only be
1
0
enantioselectivity. The different behaviour of wild-type and Beside stereoelectronic effects involved in the enzymatic
mutated transaminases was assessed by in silico substrate binding interactions, water solubility of hydrophobic substrates may also
mode studies.
strongly influence the reactivity.
Therefore, in attempt to characterise the electronic behaviour of
1
1
the amino transferase from Halomonas elongata (HEWT), we
investigated the role of different substituents on aromatic ketones,
aldehydes, and the corresponding (S)-amines. The effect on the
catalytic efficiency of the wild-type enzyme was initially studied
with different mono-substituted acetophenones, having both
electron withdrawing and donating groups (Table 1, column 3),
where L-alanine was used as amino donor. Wild-type HEWT
generally resulted poorly active, showing significant conversions
only with meta- and para-nitroacetophenones. The reasons for this
limited reactivity can be related to unfavourable electronic effects
for substrates with EDG groups, whereas only strong EWG groups
allowed significant yields by favouring the reactivity of the amine of
PMP towards the carbonyl group (Fig. 1). The absence of reactivity
towards ortho-nitroacetophenone can be ascribed to steric effects,
as previously reported for the lack of activity towards these
Introduction
The remarkable work of Codexis-Merck in evolving an amino
1
transferase to achieve
a “greener” synthesis of Sitaglipin,
highlighted the promising features of this class of enzymes as
2
biocatalytic tool for the manufacturing of chiral amines, valuable
3
,4
building blocks for a wide range of bioactive compounds. The
mechanism underlying ω-transaminase activity has been generally
5
,6
assumed similar to that of aspartate transaminase; only recently
the reaction mechanism of Chromobacterium violaceum ω-
transaminase has been investigated by means of density functional
theory calculations and the energy profile for the conversion of (S)-
1-phenylethylamine to acetophenone has been calculated. In this
study the involved transition states and intermediates have been
7
characterised. The first step in the conversion of carbonyls into
1
2
substrates with the yeast P. glucozyma ketoreductase KRED1-Pglu.
amines involves the nucleophilic attack from the amino donor
intermediate pyridoxamine-5'-phosphate (PMP) to the amino
7
acceptor carbonyl group. The stereoelectronic features of the
substrate play a crucial role; in the case of ketones, especially
aromatic ones, the carbonyl carbon is often not sufficiently
Fig. 1 Representation of the electronic density with EWG and EDG
substituted molecules.
a.
UCD School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland
Department of Food Environmental and Nutritional Sciences(DeFENS), Università
degli Studi di Milano, Via Mangiagalli 25, 20133, Milan, Italy E-mail:
francesco.molinari@unimi.it
b.
c.
School of Chemistry, University of Nottingham, University Park, Nottingham, NG7
2RD, UK E-mail: Francesca.paradisi@nottingham.ac.uk
M. Contente and M. Planchestainer contributed equally to this paper.
‡
Results and discussion
Electronic Supplementary Information (ESI) available: [Expression and purification
of HEWT, HEWT mutant generation, ketones and aldehydes, amines reactions,
chirality evaluation, transaminases’ stability, docking energy results]. See
DOI: 10.1039/x0xx00000x
An in silico model of the HEWT was generated to aid with the design
13
of key mutations, specifically introduced to probe the steric effect
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of the substituents. The three-dimensional structure was simulated Ortho- meta- and para-nitroacetophenones were selected to map
on the resolved structure of the homologs amino transaminase in-silico the active site of HEWT (Fig. 2). The steric bulk of the nitro
1
4
from Cromobacterium violaceum. Due to the medium-high group, when positioned in ortho and meta, appeared to interfere
homology between the two proteins (56%), the built model was with tryptophan 56 and tyrosine 149 in the large pocket, as well as
deemed reliable providing a quality standard score (Z score < 2.5) with phenylalanine 84 which is located between the two pockets;
1
5
and it allowed for an accurate evaluation of the catalytic pocket.
(Fig 2 A-B). On the other hand, the para position is less hindered
The enzyme topology for this class of enzymes has been well and the model predicts major interference only with W56
elucidated and it can be easily described by two pockets positioned deep into the pocket (Fig.2 C).
1
6,17
surrounding the cofactor pyridoxal phosphate (PLP).
The large
one accepts bulky groups such as aromatic rings, while the second,
smaller one, accommodates short hydrophobic groups.
Fig. 2 Model of the catalytic site of HEWT with ortho- meta- and para-nitroacetophenone.
O
NH2
0.1 mg/mL HEWT; L-Ala 1 M
X
X
PO
4
buffer 50 mM pH 8;
.1 mM PLP; 37 °C
0
10 mM
Entry
Substrate
Acetophenone
p-NO2
m-NO2
o-NO2
p-F
WT
5.0 ± 0.4
19.6 ± 0.2
10.2 ± 0.8
< 5
ee %
>99
>99
>99
W56G
ee %
Y149F
ee %
F84A
ee %
>99
95
I258A
5.0 ± 0.4
19.1 ± 0.9
8.6 ± 0.2
< 5
ee %
>99
>99
95
1
a
b
6.0 ± 0.1
15.7 ± 0.5
10.7 ± 0.7
15.9 ± 1.6
< 5
>99
96
< 5
14.2 ± 1.2
7.3 ± 0.5
< 5
5.0 ± 0.3
1
97
97
12.4 ± 0.5
6.2 ± 0.6
< 5
1
c
95
98
96
1
d
e
1
< 5
< 5
< 5
< 5
1
f
m-F
< 5
< 5
< 5
< 5
< 5
1
1
g
o-F
p-CF3
< 5
< 5
<5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
h
1
i
m-CF3
< 5
< 5
< 5
< 5
< 5
1
j
o-CF3
< 5
< 5
< 5
< 5
< 5
1
k
p-OCH3
m-OCH3
o-OCH3
p-CH3
< 5
< 5
< 5
< 5
< 5
1
l
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
1
m
1
n
o
p
< 5
< 5
< 5
1
1
m-CH3
o-CH3
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
Table 1. Molar conversions of ammination of mono-substituted acetophenones 1a-p (10 mM) with transaminases (0.1 mg/mL) in phosfate buffer medium
0 mM pH 8, DMSO 10% as cosolvent, 1 M L-Alanine and 0.1 mM PLP. Biotransformations were carried out for 24 h at 37°C under gently agitation.
5
An in silico mutagenesis approach was used to evaluate optimal accommodation with a greater effect on the ortho- substitution
1
8
substitutions. W56G on the large pocket, and F84A near the small (ESI, Table S.2); Y149A also was indicative of a better docking,
one, yielded a lower docking energy favouring the substrates however this amino acid is involved in PLP stacking, therefore a
2
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more conservative substitution (Y149F) was selected. Finally, I258 The enantioselectivity was also monitored validating that both the
appeared to be critical for enlarging the pocket; it is spatially near WT and the mutants selectively produced the (S)-amines,
W56 and prevents its side chain to rotate. A I258A mutation will confirming that the mutations did not affect the stereo-preference
allow a higher rotational flexibility to the tryptophan ring leaving of the catalyst. The W56G variant is the only one able to transform
more space for the substrate to dock (Fig. 3). Notably, the selected the ortho-nitroderivative. In fact, the replacement of tryptophan
amino acids are highly conserved among this class of amino with glycine reduced hindrance and facilitated substrate binding
transaminase (96% among 487 protein sequences aligned and Y149 (Fig 2 A). Due to the low reactivity of acetophenones, the same
19
is 100% retained).
study was performed on more reactive mono-substituted
benzaldehydes (Tab 2, entry 2a-p), using L-alanine as amino donor.
As predicted by the molecular modelling, the substitution of bulky
amino acids such as W56 and F84 with glycine and alanine
respectively, and the replacement of Y149 with phenylalanine
(
lacking the OH group fundamental for steric and electronic
interaction with the substrate), gave an enhanced molar conversion
for all the ortho-substituted aldehydes into the corresponding
primary amines with respect to the wild type transaminase.
However, in the case of meta-derivatives the activity of the
different transaminases was predominantly dependent on
substituent bulk and/or hydrophobicity whereas the electronic
properties had negligible effects. For para-substituted aldehydes,
I258A was the best variant both with EWG and EDG, as steric effects
at this position prevailed on the electronic ones; mutating I258
allowed for greater flexibility of the neighbouring W56 side chain
substantially increasing the size of the active site. The direct
mutation of W56 with glycine yielded very similar results with
respect to the wild type, highlighting the role of this amino acid in
stabilising the para-substituents. A number of chiral amines with
different substitution patterns on the aromatic ring have also been
included in this study (Tab 3, entry 3a-h) using as amino acceptor
sodium pyruvate. Amines are generally better accepted substrates
than the corresponding ketones, and steric rather than electronic
effects can easily be identified.
Fig.3. Simulation of tryptophan rotation after the substitution of
isoleucine 258 with alanine.
The four generated mutants of HEWT were thus investigated with
para- meta- and ortho- mono-substituted acetophenones as amino
acceptor (Tab. 1, columns 4-7). The biotransformations were
performed over 24 hours and under the reaction conditions
2
selected; the only substrates transformed were the NO -
acetophenones (entry 1b-d).
O
NH
2
0.1 mg/mL HEWT; L-Ala 1 M
X
X
4
PO buffer 50 mM pH 8;
0.1 mM PLP; 37 °C
10 mM
Entry
Substrate
WT
W56G
Y149F
F84A
I258A
2
a
Benzaldehyde
p-NO2
m-NO2
o-NO2
p-F
22.2 ± 1.9
72.4 ± 3.2
65.4 ± 1.3
69.2 ± 1.2
26.2 ± 1.5
64.6 ± 3.9
67.9 ± 1.5
70.8 ± 2.7
73.3 ± 2.2
90.7 ± 1.3
17.7 ± 1.7
22.6 ± 1.3
19.5 ± 0.3
14.1 ± 0.6
25.5 ± 0.8
39.6 ± 1.4
31.6 ± 0.8
70.2 ± 1.0
68.4 ± 1.1
89.3 ± 1.1
35.4 ± 1.1
79.2 ± 1.3
91.2 ± 1.4
67.0 ± 0.8
75.5 ± 0.5
99.7 ± 0.6
21.1 ± 1.2
26.9 ± 0.5
48.1 ± 0.3
18.6 ± 0.7
35.2 ± 1.0
47.1 ± 0.7
32.0 ± 0.9
59.3 ± 0.9
57.0 ± 0.8
88.8 ± 1.2
23.7 ± 0.7
80.0 ± 0.7
96.1 ± 1.8
74.5 ± 0.6
70.8 ± 1.2
98.2 ± 0.4
14.8 ± 1.5
23.5 ± 2.5
46.9 ± 0.4
17.1 ± 0.3
27.0 ± 0.2
49.9 ± 0.4
27.3 ± 0.9
56.5 ± 1.1
51.0 ± 1.9
80.6 ± 0.4
24.7 ± 0.3
76.2 ± 1.0
94.1 ± 1.0
72.9 ± 0.6
68.1 ± 0.2
94.1 ± 0.2
18.4 ± 0.7
20.3 ± 0.6
41.0 ± 0.2
20.3 ± 0.8
20.4 ± 0.6
44.4 ± 0.7
22.5 ± 0.7
80.5 ± 1.0
50.6 ± 1.2
64.8 ± 1.5
52.0 ± 1.3
65.3 ± 1.0
90.6 ± 0.7
97.5 ± 1.4
66.7 ± 1.3
92.1 ± 0.2
38.1 ± 0.2
24.1 ± 0.6
20.6 ± 0.4
28.5 ± 0.5
26.9 ± 1.7
36.5 ± 1.6
2b
2
c
2
d
e
2
2
f
m-F
o-F
2g
2h
p-CF3
2
i
j
m-CF3
o-CF3
2
2k
p-OCH3
m-OCH3
o-OCH3
p-CH3
m-CH3
o-CH3
2
l
2
m
2
n
o
p
2
2
Table 2. Molar conversions of ammination of mono-substituted benzaldehydes 2a-p (10 mM) with transaminases (0.1 mg/mL) in phosfate buffer medium 50
mM pH 8, DMSO 10% as cosolvent, 1 M alanine and 0.1 mM PLP. Biotransformations were carried out for 24 h at 37°C under gently agitation
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NH
2
O
0.1 mg/mL HEWT; Pyr 10 mM
X
X
PO
4
buffer 50 mM pH 8;
.1 mM PLP; 37 °C
0
1
0 mM
Entry
Substrate
(S)-PEA
WT
W56G
Y149F
F84A
I258A
3
a
b
99.9 ± 0.2
99.9 ± 0.1
99.9 ± 0.2
82.9 ± 2.0
99.3 ± 0.6
97.9 ± 1.0
71.7 ± 1.9
99.7 ± 0.3
99.7 ± 0.3
95.4 ± 1.3
95.5 ± 0.2
84.6 ± 1.4
98.0 ± 1.0
94.8 ± 0.9
78.0 ± 1.1
98.6 ± 1.5
99.5 ± 0.5
95.4 ± 0.1
91.7 ± 1.4
68.3 ± 1.2
97.9 ± 0.6
97.9 ± 0.3
46.2 ± 2.4
97.3 ± 0.8
99.6 ± 0.2
47.7 ± 1.3
34.8 ± 1.9
12.8 ± 0.1
70.7 ± 0.6
75.3 ± 0.2
10.3 ± 1.0
76.7 ± 0.6
83.5 ± 1.0
95.9 ± 0.4
93.2 ± 2.5
20.2 ± 0.1
98.3 ± 0.1
98.2 ± 0.6
72.1 ± 1.2
98.4 ± 0.1
3
(S)-p-F
3
c
(S)-m-F
3
d
e
(S)-o-F
3
(S)-p-OCH3
(S)-m-OCH3
(S)-o-OCH3
(S)-p-CH3
3
f
3g
3h
Table 3. Molar conversions of oxidation of mono-substituted amines 3a-h (10 mM) with transaminases (0.1 mg/mL) in phosfate buffer medium 50 mM pH 8,
DMSO 10% as cosolvent, 10 mM pyruvate and 0.1 mM PLP. Biotransformations were carried out for 24 h at 37°C under gently agitation
Interestingly, among the mutants, only W56G had better activity exploiting the QuickChange Primer Design tool (Agilent
with the ortho-substituents (o-F- and o-OCH
3
-amine), while the Technologies®) were synthesised from Eurofins Genomics®. The
other mutations showed a major destabilizing effect. The molar four HEWT mutants were achieved using the following primers:
conversion of F84A indeed dropped to just over 10% conversion W56G
5'-cggcatggccgggcttggctgcgtgaatctc-3';
F84A
Y149F
5'-
5'-
with both substrates in the same reaction time. This could be ctgccgtactacaacaccgccttcaagaccacgcatcc-3';
attributed to an impairment effect of the first step in the catalytic gtcgcgagaacgcctttcacggctcca-3';
I258A
5'-cgccgacgaggt
cycle where the substrate displaces the catalytic lysine from the ggcctgcggcttcggg-3' (mutated codon is underlined).
PLP. F84 appears essential in the case of substituted aromatic
Ketones and aldehydes reactions
amines in stabilising the binding of the substrate as well as assisting
The batch reactions to evaluate the conversion after 24 h, with
wild-type and mutated HEWT, were performed in 1.5 mL micro
centrifuge tubes; 200 µL reaction mixture in 50 mM phosphate
buffer pH 8.0, containing 10 mM amino acceptor substrate
dissolved in 10% DMSO, 1.0 M L-Alanine (amino donor substrate),
the catalytic mechanism. However, this effect is not observed with
the natural substrate (S)-PEA probably due to a very efficient
reaction that masks the negative impact of the mutation. These
data were also confirmed by very different K
W56G and F84A compared with wild type (0.61 o-FW56G; 1.80 o-
F84A; 1.38 o-FWT and 0.45 o-OCH3 W56G; 3.97 o-OCH F84A; 1.98 o-
m
for o-amines with
0
.1 mM PLP, and 0.1 mg/mL enzyme, was left under gentile shaking
at 37 °C. 10 µL aliquots were quenched with trifluoroacetic acid
TFA) 0.2% at 24 hours and then analysed by HPLC equipped with a
F
3
OCH3 WT expressed in mM). W56G and I258A, predicted as the best
mutated transaminases for para-derivatives, in oxidising conditions
showed very similar results with respect to wild type enzyme. The
mutations can effectively alter the efficiency of the enzymes in just
one direction, therefore also altering the equilibrium of the
reaction.
(
Supelcosil LC-18-T column (250 mm x 4.6 mm, 5 µm particle size;
Supelco, Sigma-Aldrich, Germany). The compounds were detected
using an UV detector at 210 nm, or 250 nm after an isocratic run
with 25% acetonitrile/75% water with TFA 0.1% v/v at 25 °C with a
flow rate of 1 mL/min. The retention times in minutes are listed in
table S.1. Calibration curve for substrate and product were carried
out with commercial standard and the conversion was then
estimated, when possible, both from the substrate signal
decreasing and product signal forming.
Experimental
Expression and purification of the HEWT in E. coli
Protein expression and purification was performed following
1
1
Amines reactions
previously reported protocols. The proteins yields after the IMAC
purification were estimate to be: WT 100 mg, W56G 93 mg, F84A The batch reactions to evaluate the conversion after 24h, with wild-
7 mg, Y149G 32 mg, and I258A 88 mg for 1 Liter of expression type and mutated HEWT, were performed in 1.5 mL micro
4
2
0
media. The assay derived from Schatzle, et al. was used as centrifuge tubes; 200 µL reaction mixture in 50 mM phosphate
standard enzymatic assay following the procedure applied in Cerioli, buffer pH 8.0, containing 10 mM amino donor substrate dissolved
11
et al. to assess the enzymes activity in standard conditions and in 10% DMSO, 10 mM Pyruvate (amino acceptor substrate), 0.1 mM
evaluate then their stability (see ESI).
PLP, and 0.1 mg/mL enzyme, was left under gentile shaking at 37 °C.
The conversion was evaluated by HPLC with the same procedures
described above. Retention times are listed in Table S.1 in ESI. The
kinetic profiles for the amine substrates were measured using a Bio-
Tek EPOC2 microplate reader. The reaction mixture was set up in
HEWT mutant generation
The SupC gene of HEWT harboured in a pRSETb plasmid was
mutated using the QuikChangeXL single point mutation kit provided
by Agilent Technologies®. The oligonucleotide primers designed
3
00 ꢀL of activity buffer (50 mM phosphate buffer pH 8.0)
4
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containing a variable concentration of substrate amino donor (0.01 of homologs ω-transaminases. Therefore, as general rule, the
to 2 mM), 10 mM of pyruvate, 0.1 mM PLP and an appropriate mutation of large pocket tryptophan with a smaller residue yields to
an improvement in the reactivity of ortho-substituted aromatic
compounds. The change of the large pocket isoleucine provides
amount of enzyme. The reaction rate was detected at 245 nm over
2
0 minutes at 25°C. The molar extinction coefficient was calculated
for each product in order to calculate the reaction rate in mM/s
data not shown). The data were elaborated by fitting them with
instead an increase in activity of para-substituted molecules. On the
other hand, F84 between the two pockets was very sensible to
substitution and when it was changed for smaller alanine, it gave a
worst conversion of ortho and para aromatic amines. Consequently,
this position has a fundamental role in the oxidation step of amino
transaminase and it should be preserved in order to maintain the
optimal enzyme efficiency.
(
the Lineweaver–Burk double reciprocal plot which yielded the
Michaelis-Menten parameters (Km and Kcat).
Chirality evaluation
To evaluate the enantioselectivity of the enzymes, the chiral amine
products were assessed by chiral HPLC analysis, following
derivatization to the corresponding amides by adapting the method
Acknowledgements
®
reported by Andrade et al. with Codex ATA Screening Kit. Addition
The authors wish to thank Cariplo Foundation for funding the
project “INnovative Biocatalytic OXidations – INBOX” (Rif.2014-
of 100 µL 5 M NaOH to the sample was followed by extraction in
900 µL methyl-tert-butyl ether (MTBE) and then by addition of 5 µL
0
568) “Ricerca integrata biotecnologie industriali” (Dr. Contente)
triethylamine and 5 µL acetic anhydride. A normal-phase HPLC and Science Foundation Ireland through the Synthesis and Solid
method was performed on an Agilent Technologies 1200 equipped State Pharmaceutical Centre grant number 12/RC/2275
®
(Planchestainer and reagents).
with a Chiralpak IC (250mm x 4.6 mm, 5 µm particle size).
Detection at 254 nm after an isocratic run 95:05 Heptane:Ethanol at
2
5 °C with a flow rate of 1 mL/min. Employing this method, product
Notes and references
and starting materials were separated with the following retention
times (in minutes): acetophenone (7.1), derivatized (R)-1-
phenylethylamine (16.7) and (S)-1-phenylethylamine (19.1), 4-
nitroacetophenone (12.4), derivatized (R)-4-nitrophenylethylamine
1
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. Huisman, G. J. Hughes, Science, 2010, 329
305.
,
(
22.4) and (S)-4-nitrophenylethylamine (24.2), 3-nitroacetophenone
13.6), derivatized (R)-3-nitrophenylethylamine (23.7) and (S)-3-
(
2
3
4
5
6
7
D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell and W.
Kroutil, Adv. Synth. Catal., 2008, 350, 2761.
nitrophenylethylamine
(26.7),
2-nitroacetophenone
(14.2),
derivatized (R)-3-nitrophenylethylamine (24.8) and (S)-3-
nitrophenylethylamine (27.1) .
J. Rudat, B. R. Brucher and C. Syldatk, AMB Express, 2012,
1.
2,
1
Protein modelling and structural analysis
The protein model was build using the SWISS-MODEL Homology
D. Koszelewski, K. Tauber, K. Faber, W. Kroutil, Trends
Biotechnol., 2010, 28, 324.
1
5
Modeling tool, superimposing the HEWT protein sequence on the
resolved structure of the homologues amino transaminase from
14
K. E. Cassimjee, B. Manta and F. Himo Org. Biomol. Chem.,
015, 13, 8453.
Cromobacterium violaceum (PDB: 4a72). Substrate docking was
2
2
1
performed exploiting the open source tool Autodock Vina. The
results elaboration and the structural evaluation was achieved using
the molecular visualization system PyMOL (open source license).
Sequence analysis and alignment was performed with CunSurf,
G. D. Smith, R. Harrison and R. Eisenthal, Neurochem. Res.,
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2
2
J. F. Kirsch, G. Eichele, G. C. Ford, M. G. Vincent, J. N.
Jansonius, H. Gehring and P. Christen, J. Mol. Biol., 1984,
software for multiple blast provided by BioSoft.
1
74, 497.
Conclusions
8
9
J. S. Shin and B. G. Kim, J. Org. Chem., 2002, 67, 2848.
Transaminase from Halomonas elongata (HEWT) showed a wide
variety of catalytic abilities towards different aromatic substrates
D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz, D.
Rozzell and W. Kroutil, Angew. Chemie - Int. Ed., 2008, 47
337.
,
(
ketones, aldehydes, amines). Acetophenone derivatives were poor
substrates for the wild-type enzyme, since only meta- and para-NO
acetophenones were reduced with molar conversions ranging from 10 E. S. Park, S. R. Park, S. W. Han, J. Y. Dong and J. S. Shin, Adv.
0 to 20%. Four mutants were designed following in silico
Synth. Catal., 2014, 356, 212.
substrate-enzyme binding mode studies. Mutant W56G proved able
9
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1
to catalyse also the amination of ortho-NO
2
acetophenone, as a 11 L. Cerioli, M. Planchestainer, J. Cassidy, D. Tessaro and F.
Paradisi, J. Mol. Catal. B Enzym., 2015, 120, 141.
result of reduced interferences and facilitated substrate binding.
Variants W56G and F84A, which have an enlarged binding pocket,
showed better performances with ortho-substituted benzaldehyde
derivatives, whereas I258A was the best mutant for para-
derivatives. Since the investigated mutations involved highly
conserved amino acids, these findings might reflect the behaviour
1
2 M. L. Contente, I. Serra, L. Palazzolo, C. Parravicini, E.
Gianazza, I. Eberini, A. Pinto, B. Guidi, F. Molinari, D.
Romano, Org. Biomol. Chem, 2016, 14, 3404.
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DOI: 10.1039/C6OB01629D
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