Expanding the substrate scope of phenylalanine ammonia-lyase from: Petroselinum crispum towards styrylalanines

Article abstract of DOI:10.1039/c7ob00562h

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d-which are less studied and synthetically challenging unnatural amino acids-by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower k_cat/K_M value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL-as indicated by molecular modeling-might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

Full text of DOI:10.1039/c7ob00562h

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Organic & Biomolecular Chemistry
ARTICLE
Expanding the substrate scope of phenylalanine ammonia-lyase
from Petroselinum crispum towards styrylalanines
Received 00th January 20xx,
Accepted 00th January 20xx
László Csaba Bencze,§a Alina Filip, §a Gergely Bánóczi,b Monica Ioana Toșa,a Florin Dan Irimie,a
Ákos Gellért,c László Poppe,*a,b,d and Csaba Paizs*a
DOI: 10.1039/x0xx00000x
This study focused on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum
(PcPAL) towards the L-enantiomers of racemic styrylalanines rac-1a-d – which are less studied and synthetically
challenging unnatural amino acids – by reshaping the aromatic binding pocket of the active site of PcPAL by point
mutations. Ammonia elimination from L-styrylalanine (L-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) happened with
777-fold lower kcat/KM value as the deamination of the natural substrate, L-Phe. Computer modeling of the reactions
catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental
interactions between the aromatic moiety of L-styrylalanine, L-1a, and the phenyl ring of F137 residue in the aromatic
binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues, resulted a small mutant
library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform L-styrylalanine with comparable
activity that of the wt-PcPAL with L-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia
elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to D-1a-d by kinetic resolution,
even though the D-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL
towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling indicating the more relaxed enzyme-
substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons.
Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed both with wt-PcPAL and F137V-PcPAL.
The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and non-productive
binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL – as indicated by
molecular modeling – might be responsible for inactivity of the PcPAL variants in the reverse reaction. Modeling predicted
the F137V mutation beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR
process of 4-triflouromethylstyrylalanine.
www.rsc.org/
attention.4- 7 An attractive enzymatic route for the synthesis of
non-natural phenylalanine analogues is provided by aromatic
ammonia-lyases and aminomutases which show high
structural and sequence similarities, and have a common
electrophilic prosthetic group, the autocatalytically forming
3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO).8- 12
Recent advances in improvement of the functional properties
of the MIO-enzymes initiated their application as biocatalysts
for the synthesis of non-natural amino acids, well exemplified
by the phenylalanine ammonia-lyase (PAL)-based process,
developed by DSM (Netherlands), for the multiton scale
production of (S)-2,3-dihydro-1H-indole-2-carboxylic acid.13
Due to its broad substrate tolerance, phenylalanine ammonia-
lyase from Petroselinum crispum (PcPAL) was one of the most
frequently used aromatic amino acid ammonia-lyase applied
as biocatalyst.14,15,16 Immobilization of isolated PcPAL led to
increased operational stability and enabled the employment of
PcPAL for the synthesis of non-natural substrates in
continuous-flow packed-bed reactors,17 in magnetic
nanoparticle-filled microfluidic systems,18,19 and provided new
Introduction
The synthesis of unnatural aromatic amino acids in
enantiopure form is still a challenge of synthetic chemistry,
highlighted by the significant interest towards these building
blocks in the development of therapeutic peptides and
proteins.1- 3
Since phenylalanine – as a pharmacophore element – has an
important role in biologically active peptides and proteins,
development of synthetic methods leading to sterically more
demanding analogues of this amino acid has gained increased
a.Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and
Chemical Engineering, Babeș-Bolyai University of Cluj-Napoca, Arany János Str.
11, RO-400028 Cluj-Napoca, Romania. E-mail: paizs@chem.ubbcluj.ro
b.Department of Organic Chemistry and Technology, Budapest University of
Technology and Economics, Műegyetem rkp. 3, H-1111 Budapest, Hungary. E-
mail: pope@mail.bme.hu
c. Agricultural Institute, Centre of Agricultural Research, Hungarian Academy of
Sciences, Brunszvik u. 2, H-2462 Martonvásár, Hungary.
d.SynBiocat Ltd. Lövőház u. 19/1, H-1024 Budapest, Hungary.
§ LCB and AF contributed equally to this work.
*Co-corresponding authors.
Electronic Supplementary Information (ESI) available on experimental details. See
DOI: 10.1039/x0xx00000x
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perspectives in the development of stable and efficient acid (2a) as the major product.28 Considering the high
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DOI: 10.1039/C7OB00562H
structural and sequence similarities between TcaPAM and
catalysts for industrial applications.
Numerous evidences suggest that interactions between the PcPAL, it seemed commendable to explore the potential of
amino group of the substrate L-Phe and the electrophilic MIO PcPAL in the kinetic resolution of α-styrylalanines and
of PAL facilitate the ammonia-lyase reaction.20 The crystal stereoselective amination of (2E,4E)-styrylacrylates in the
structures of phenylalanine 2,3-aminomutases from Pantoea reverse reaction (Scheme 1).
agglomerans (PaPAM) complexed with
from Taxus chinensis (TchPAM) complexed with
-phenylalanine,22 the tyrosine 2,3-aminomutase from analogues – created mutant variants of recombinant PcPAL
Streptomyces globisporus (SgTAM) co-crystallized with with enlarged aromatic binding pocket enabling smooth fit of
difluoro-
-tyrosine,23 all with a covalent bond between the styrylalanines with increased distance between the aromatic
L
-phenylalanine21 and The present study – with the aim of extending the substrate
L-α,α-difluoro- scope of PcPAL towards sterically demanding phenylalanine
β
L-α,α-
β
amino group of the substrate/inhibitor and the exocyclic moiety and the α-stereogenic carbon into the active site.
methylene carbon of the MIO supported a reaction mechanism The results of this study were rationalized by molecular
proceeding through the so called N-MIO intermediate. This modeling in the framework of a straightforward physics-based
was also in agreement with QM/MM calculations on TAL24 and approach
based
on
ligand/N-MIO
intermediate
PAL19 supporting ammonia elimination via an N-MIO poses/conformation sets and several molecular descriptors to
intermediate and suggesting the formation of similar N-MIO approximate changes on the potential energy surface and thus
complexes as a common feature of the mechanisms for all characterize certain phenomena of the catalysis.
MIO-enzymes.20
Proceeding from the N-MIO intermediate of PAL by the aid of
the catalytically essential Tyr110,25 the elimination is either
stepwise i) through a benzylic carbanion (E1cB), ii) or through a
carbocation (E1), or iii) it is a concerted process (E2). A
thorough kinetic analysis of the PAL reaction,26 based on
kinetic isotope effects of [15N]- and/or [3-2H2]-phenylalanine
favored the mechanism via a carbanionic intermediate (E1cB).
In a recent study,27 a previously unrevealed, competing MIO-
independent reaction pathway was reported with PALs from
Anabaena variabilis (AvPAL), Rhodotorula glutinis (RgPAL), and
parsley (PcPAL) which proceeds in a non-stereoselective
Results and discussion
Initial studies and F137X-PcPAL mutant set construction
First, to assess the applicability of wt-PcPAL to the
styrylalanine compound family, the commercially available
L-
styrylalanine ( 1a) was tested as substrate in the ammonia
L-
elimination reaction (Scheme 1), monitored by HPLC and UV-
based assays. The 1H-NMR spectra of the reaction mixtures
confirmed the formation of (2E,4E)-5-phenylpenta-2,4-dienoic
acid
elimination catalyzed by wt-PcPAL showed 14.7-fold lower kcat
and 777-fold lower kcat/KM values for the deamination of 1a
in comparison with the deamination of the natural substrate,
-Phe (Table 1).
The low enzyme activity, observed with
(2a) as product. Kinetic studies of the ammonia
manner and results in the generation of both L- and D-
phenylalanine derivatives. Isotope-labeling studies and
mutagenesis of key active site residues were consistent with
L-
amino acid deamination occurring by
elimination mechanism in this case as well.
a stepwise E1cB
L
L-styrylalanine (L-1a),
It was already shown that phenylalanine 2,3-aminomutase
was rationalized by molecular modeling. Evaluation of
energetics indicated as the most disadvantageous interaction
from Taxus canadensis (TcaPAM) could transform (S)-α-
styrylalanine (L-1a) into (2E,4E)-5-phenylpenta-2,4-dienoic
COOH
NH2
COOH
COOH
NH2
PcPAL
+
A
Aryl
Aryl
Aryl
8.8
Tris,
pH
-
NH3
-
-
1a-d
rac
1a-d
2a-d
D
COOH
COOH
PcPAL
B
Aryl
Aryl
6M NH
10
3, pH
NH2
-
2a-d
L
1a-d
a
c
b
d
Cl
Aryl:
Cl
N
O2
Scheme 1 The (A) ammonia elimination from racemic styrylalanines 1a-d and the (B) attempted ammonia addition onto styrylacrylates 2a-d catalyzed
2 | Org. Biomol. Chem., 2017, 00, 1-9
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Table 1 Kinetic parameters for wt-PcPAL and for F137X-PcPAL mutants in ammonia elimination from L-phenylalanine and from L-styrylalanine (L-1a)
PcPAL
L-Phenylalanine
kcat (s-1)
0.694 ± 0.020
0.173 ± 0.001
0.283 ± 0.001
0.052 ± 0.003
L-Styrylalanine
kcat (s-1)
0.0471 ± 0.0003
0.4220 ± 0.0280
0.1320 ± 0.0026
0.0345 ± 0.0038
KM (μM)
83 ± 5
86 ± 10
1732 ± 15
4969 ± 153
kcat/KM (M-1 s-1)
8361 ± 291
2011 ± 131
163 ± 2
KM (μM)
4384 ± 158
186 ±6
1173 ± 70
4120 ± 270
kcat/KM (M-1 s-1)
10.7 ± 0.4
2269 ± 168
112.5 ± 7.2
8.3 ± 0.2
wt
F137V
F137A
F137G
10.4 ± 0.9
Table 2 Kinetic parameters for wt- and F137V-PcPALs in the ammonia elimination of racemic styrylalanines rac-1a-d
Substrate
wt-PcPAL
F137V-PcPAL
KM (μM)
kcat (s-1)
kcat/KM (M-1 s-1)
KM (μM)
kcat (s-1)
kcat/KM (M-1 s-1)
rac-1a
rac-1b
rac-1c
rac-1d
395 ± 6
154 ± 7
28 ± 1
0.00620 ± 0.00070
0.00024 ± 0.00001
0.00034 ± 0.00002
0.00320 ± 0.00030
15.6 ± 0.9
1.5 ± 0.03
12.1 ± 0.6
11.1 ± 0.5
201 ± 12
78.3 ± 2
94.7 ± 2
326 ± 10
0.2760 ± 0.0130
0.0786 ± 0.0044
0.1570 ± 0.0020
0.0099 ± 0.0001
1373 ± 97
1004 ± 86
1657 ± 10
30.2 ± 1.9
287 ± 3
the one between the phenyl ring of the N-MIO intermediate reaction rate might be improved by reducing the clash
from 1a and the phenyl ring of residue F137 in the aromatic between the aromatic moieties of 1a in its N-MIO state and
L-
L-
binding pocket of wt-PcPAL (Figure 1). Moreover, enumeration F137 within the active site of wt-PcPAL, and simultaneously by
of the possible conformations indicated three major species. In relocating the population of the unproductive conformation.
panel
A of Figure 1, two apparently catalytically active Notable, that earlier, in case of 4-substituted cinnamates as
conformations, resembling the active arrangement of substrates in the reverse amination reaction with PcPAL,
29
phenylalanine in PcPAL,19 are depicted. These two N-MIO mutations of the F137 site were efficacious
.
Therefore, to
intermediate conformations can result in two different provide more space for the phenyl ring of 1a, residue Phe137
L-
arrangements of the diene product (2a), the s-cis [referred as in the hydrophobic pocket of wt-PcPAL was changed to smaller
pro-s-cis (psc)] or the s-trans [referred as pro-s-trans (pst)] hydrophobic residues Val, Ala, and Gly in mutants F137V-,
conformations. Panel
conformation adopting a somewhat different arrangement of the wild-type and the mutant PcPALs were determined in
with significantly less stress exerted by F137. However, this the ammonia elimination reaction for the natural substrate
specimen would lead to (2Z,4E)-5-phenylpenta-2,4-dienoic Phe and for 1a (Table 1).
acid which was not detected by NMR in the reaction mixture. The ratio of kcat and KM – often referred as the “catalytic
Thus, conformation 1au could be considered as an efficiency” – is a useful index for comparing the efficiency of an
unproductive state. These modeling results implied that the enzyme acting on alternative substrates
B of Figure 1 depicts a third F137A-, and F137G-PcPAL, respectively. The kinetic parameters
L-
L-
L-
30
.
Not surprisingly, all
A
B
Figure 1 Active site models of the three major conformations of the N-MIO complex of L-1a with wt-PcPAL [colors refer to the atomic contributions to
the total energy ranging from green (beneficial, ≤-5 kcal mol-1) to red (detrimental, ≥2 kcal mol-1)]. (A) The two allegedly catalytically active
conformations L-1apsc and L-1apst leading to the product 2a in s-cis (synperiplanar) or s-trans (antiperiplanar) arrangements, respectively. (B) the
apparently catalytically inactive conformation L-1au compared to the apparently catalytically active L-1apsc conformation.
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mutants showed decreased – declining with side chain volume Supplementary Information, ESI) and tested with VFie1w3A7rVtic-lPe cOPnlAinLe
– catalytic efficiencies in the ammonia elimination reaction of and wt-PcPAL as biocatalysts (Scheme 1)D. OI: 10.1039/C7OB00562H
the natural substrate,
However, it can be clearly seen that F137V has superior
catalytic efficiency with 1a as substrate, with the nearly 240- F137V-PcPAL. However, the F137V mutant showed
fold increased kcat/KM value compared to wt-PcPAL. The F137A significantly mproved catalytic activities (kcat and kcat/KM) in
mutation also provided an increased kcat/KM value with 1a comparison with the wild-type enzyme in all cases (Table 2).
(10.5-fold enhancement), while the F137G mutation showed The synthetic usefulness of F137V-PcPAL towards
no beneficial effect on the transformation of 1a styrylalanines in the ammonia elimination reactions was
L
-Phe
,
in comparison with wt-PcPAL. In the ammonia elimination reaction all styrylalanines (rac-1a-
d) were accepted as substrates by wt-PcPAL as well as by
L-
i
L-
L-
.
Interestingly, kcat values showed also substantial variances. The further tested by kinetic resolutions (KRs) with 0.1 mmol rac-
trends and maxima were almost the same as in the case of the 1a-d at 5 mM concentration using 0.5-1.0 mg of pure PcPALs.
kcat/KM values. Regarding
side chain volume, except for a surprising spike with F137A- styrylalanines rac-1a-d (Figure 3), and the enantiomeric excess
PcPAL. Similarly, F137V-PcPAL with 1a showed a tremendous, of the residual substrates 1a-d were both monitored (Table
L-Phe, kcat declined with decreasing The conversion of the PcPAL-catalyzed reactions from racemic
L-
D-
almost 9-fold increase; F137A showed only a moderate 2.8- 3). In case of longer reaction times (>48 h), a fresh enzyme
fold increase; and F137G showed no improvement in kcat batch was added to the reaction mixture at the end of each 48
compared to wt-PcPAL.
h period to overcome enzyme inactivation during long
A comparison of the covalent pro-s-cis intermediates from L-1a incubation times.
within wt-PcPAL and F137V-PcPAL is shown in panel
A
of Time course analysis of the conversion of the KRs of rac-1a-d
Figure 2. The aromatic moiety of the N-MIO intermediate from (Figure 3) also indicated the beneficial catalytic properties of
1a is forced towards I460 within the active site of wt-PcPAL, F137V-PcPAL compared to that of wt-PcPAL. The theoretically
L-
unlike in the case of the more relaxed complex within F137V- possible conversion in a kinetic resolution (KR) with exclusive
PcPAL, meanwhile the alanyl substructure of the substrate – selectivity (50%) could be almost perfectly approached (c
which is directly involved in bond rearrangement – is kept >49%) with the F137V mutant in reactions of rac-1a,c after
relatively unchanged in both cases. It is worth noting that upon moderate reaction times (24 h and 134 h, respectively) and in
binding of
undergoes conformational change at the I460-side chain which 300 h, respectively). The unusually long time required for the
reshapes the active site and alters the energetics of distinct compounds rac-1b can be significantly shortened by using
L-1a to wt-PcPAL the active site supposedly the cases rac-1b,d after longer incubation times (274 h and
,d
substrate conformations.
higher enzyme to substrate ratio. This might be easily
performed by applying immobilized forms of PcPAL in
microreactors.17
Synthetic applications in kinetic resolution
On the other hand, in reactions of racemic styrylalanines rac-
Next, we wanted to explore the potential of F137V-PcPAL in
the synthetically valuable ammonia elimination and addition
reactions of various styrylalanines and their acrylic
counterparts. Therefore, racemic styrylalanines rac-1a-d as
well as the corresponding styrylacrylates 2a-d were
1a-d with wt-PcPAL, almost complete conversion of the L-
enantiomer could be obtained only with rac-1a (c >49% after
274 h), while the conversions of rac-1b-d could not approach
the theoretically possible value within 500-600 h.
KRs of rac-1a-c with wt-PcPAL showed that enantiomeric
A
B
Figure 2 Molecular models of various N-MIO intermediates in PcPALs. (A) Arrangement of covalent intermediate L-1apsc in wt-PcPAL (relevant residues
are in red) and in F137V-PcPAL (relevant residues are in cyan). Note the different side chain conformations of I460. (B) Arrangement of the
unproductive covalent complex from D-1a (yellow) compared to the productive intermediate L-1apsc (red) in wt-PcPAL.
synthesized (see Schemes S1 and S2 in Electronic excess of residual D-1a-c fractions were in close agreement
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Table 3 Kinetic resolution of styrylalanines rac-1a-d and enantiomeric excess of the residual D-1a-d in the ammonia elimination reactions catalyzed by
PcPAL variants.
Entry
PcPAL
F137V
Substrate
rac-1a
rac-1b
rac-1c
rac-1d
rac-1a
rac-1b
rac-1c
rac-1d
Time (h)
24
c (%)
50
50
50
50
50
29
36
31
Eetheor (%)a
100
100
100
100
100
41
Eeobs (%)
>99
>99
>99
>99
>98
41
1
2
3
4
5
6
7
8
274
134
300
274
504
600
600
wild-type
56
45
55
41
a Eetheor= c/(1-c) for a fully selective kinetic resolution
rac-1a F137V
rac-1a wt
rac-1b F137V
rac-1b wt
rac-1c F137V
rac-1c wt
rac-1d F137V
rac-1d wt
60
50
40
30
20
10
0
0
50
100
150
Reaction time (h)
200
250
300
Figure 3 Comparison of the time profiles of the conversions in the ammonia elimination reactions from rac-1a-d catalyzed by F137V-PcPAL (continuous
lines) and wt-PcPAL (dashed lines).
with the theoretical values of a fully selective KR indicating showed only
excellent enantiomer selectivity and preference of the process experimental errors. Because the residual fractions from the
to digest the -enantiomers, 1a-c (Table 3). Although the KRs of rac-1a-d had quite high enantiomeric excess at 50%
enantiomeric excess of the residual 1d fractions were conversion (ee >99%, see Table 3), the -enantiomers 1a-d
somewhat smaller as expected for a fully selective KR, it was were apparently not converted. This implied 1a being a
not possible to determine whether this happened due to reversible inhibitor with considerable affinity forming an
experimental error of the HPLC process or due to a non- unproductive enzyme– -substrate complex. The covalent,
perfect enantiomer selectivity of the KR from rac-1d unproductive N-MIO complex from 1a showed astonishing
KRs of rac-1a-d with F137V-PcPAL, however, showed that the overlap with the productive intermediate 1apst within the
residual 1a-d fractions had enantiomeric excess values over active site of wt-PcPAL (panel in Figure 2).
a minor variation, within the range of
L
L-
D-
D
D-
D-
D
.
D-
L-
D-
B
99% indicating unquestionably exclusive enantiomer selectivity
in all cases. This was also obvious from the time course curves
of conversions approaching asymptotically the 50% value
when F137V-PcPAL was applied as biocatalyst in the KRs
(Figure 3). Time course profiles of the reactions and the
Rationalization of the reactions of styrylalanines rac-1a-d
catalyzed by the PcPAL variants
To get a more detailed view of the catalytic process, a
quantitative model was created and validated. Because N-MIO
complexes were assumed as a common feature of the
mechanism for all MIO-enzymes,20 the enzyme-substrate
complexes within the active site of the PcPAL variants were
constructed as N-MIO intermediates of L- and D-1a-d applying
an induced-fit covalent docking protocol which was previously
used to rationalize the reactions of various substrates within
comparison of the kinetic constants with
with rac-1a (Table 2) suggests that -styrylalanine inhibits the
ammonia elimination of 1a both with wt-PcPAL and F137V-
PcPAL. If 1a had no interaction with wt-PcPAL than only a 2-
fold higher apparent KM of the racemate rac-1a would be
observed as compared to the pure -enantiomer 1a without
affecting the kcat value. However, when rac-1a was applied as
substrate instead of the pure -enantiomer 1a, the Vmax (thus
implicitly the apparent kcat value) decreased 7.6-fold, while the
M value decreased 11-fold. With F137V-PcPAL, the differences
L-1a (Table 1) and
D
L-
D-
L
L-
another MIO enzyme, the phenylalanine 2,3-aminomutase
31
L
L-
from Pantonea agglomerans
.
The non-covalent substrate-binding states of L- and D-1a-d –
directly preceding their N-MIO intermediate states – were also
K
became less significant, kcat decreased only 1.5-fold, and KM
modeled. Because the calculations did not reveal any
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Organic & Biomolecular Chemistry
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ARTICLE
Organic & Biomolecular Chemistry
unproductive states for these non-covalent complexes, they were used, one was associated with the acidity ofVtiheweAβrt-icplerOontloinne
will not be discussed hereafter.
(
cBRΔE, Table 4), and the other one waDsOaI:s1s0o.1c0ia3t9e/Cd7OwBi0th056th2He
R
The occurrence of the non-stereoselective, MIO-independent steric strain (Ster ΔE, Table 4). Applying these descriptors, Eq. 3 is
reaction pathway27 was ruled out experimentally. The derived,
attempted ammonia elimination from rac-1a-d by wt-PcPAL
#
#
L−푝s푐/푅푇+ln 푥L−푝s푐
푘cat = 푎(푒푏∗∆퐸
+ 푒푏∗∆퐸L−푝s푡/푅푇+ln 푥L−푝s푡
)
with inactivated MIO group (obtained by treatment with
sodium borohydride)32 showed no product formation. The
conversion of 4-nitrophenylalanine with the sodium
borohydride treated wt-PcPAL served as positive control for
integrity of the residual parts of the active site.
To investigate the catalytic activities quantitatively with all
substrate and enzyme mutant combinations, the apparent kcat
values – as determined from the maximum reaction rates
according to the left-hand side of Eq. 1 – were decomposed
into their constituents.
(3)
where a and b are constants, and ΔE# is the sum of Strain ΔE and
R
cBRΔE.
Non-linear estimation of the constants in Eq.
3 gave
statistically significant regression (R2=0.8, p=0.002) and
parameters (a=90.63, p=0.023; b=0.0791, p=0.014). The
complete derivation of Eq. 3 is described in the ESI).
Evaluation of the F137V mutation based on computed data
(Table 4) clearly demonstrated that the affinities of
L-
1a-d in
the pro-s-cis conformation (b ΔEL-psc) increased substantially, as
it was assumed. Moreover, the affinities of 1a-d in the pro-s-
R
At substrate saturation, when the concentration of the free
enzyme is negligible, four species of enzyme–substrate
intermediate complexes (EL-psc, EL-psc, EL-u, and ED), can be
present in thermal equilibrium (allosteric inhibition was not
L-
trans conformation increased even more, as their population
increased relative to the pro-s-cis conformation (xL-pst vs. xL-psc),
with the exception of
considered in this study). As discussed previously, from the
enantiomer two catalytically active enzyme–substrate
intermediate complexes (the s-cis and s-trans 1a-d–N-MIO
states, abbreviated as EL-psc, EL-pst) and an unproductive one
(the unproductive 1a-d–N-MIO states, abbreviated as EL-u
could be formed, while another unproductive complex could
be formed from the -enantiomers (the unproductive 1a-d
N-MIO states, abbreviated as ED). Thus, according to Eq. 1, the
L-
L-1d with F137V-PcPAL. The
unproductive complexes of 1a-d (xD and xL-u), however, showed
a more complex situation. By the F137V-mutation, in some
cases unproductive states were displaced (e.g. 1c), and in
some other cases they were introduced (e.g. 1d). These results
indicated that the simple increase of active site volume is not
sufficient from the standpoint of enzyme design, and a more
sophisticated active site shape altering scheme – taking the
relative spatial requirements of the intermediate states and
unproductive states into account – should be employed.
Inspection of the descriptors for the acidity of the pro-S β-
L-
L-
)
D
D-
–
catalytic activity is the sum of the activities of EL-psc and EL-pst
.
푉
max
= 푘cat[퐸∗] = 푘cat,L−푝s푐[퐸푝s푐] + 푘cat,L−푝s푡[퐸푝s푡
]
(1)
R
hydrogen (cB ΔE) – which was derived from single point DFT
calculations on a truncated part of the intermediate model –
Expressing the substrate concentrations as molar fractions and
substituting the Eyring equation yields Eq. 2
revealed that in almost all cases acidity of the pro-s-trans
R
conformations compared to the pro-s-cis conformations (cB ΔEL-
-
pst cBRΔEL-psc) was comparable or higher, except for 1b/wt-PcPAL
푘B푇
푘B푇
#
#
L−푝s푐/푅푇
L−푝s푡/푅푇
(2)
푘cat
=
푒∆퐺
푥
푝s푐
+
푒∆퐺
푥푝s푡
and 1d/F137V-PcPAL. Thus, the higher proportion of the pro-s-
trans conformations (xL-pst), enabling more facilitated reaction
due to the more acidic pro-S β-hydrogens, could contribute to
the increased activity of F137V-PcPAL. Moreover, the acidity
ℎ
ℎ
where T is the temperature, kB is the Boltzmann constant, h is
the Planck constant, ΔG# is the corresponding Gibbs energy of
activation, and x is the corresponding molar fraction. To
approximate the Gibbs energy of activation, two descriptors
R
R
descriptors cB ΔEL-psc and cB ΔEL-pst varied remarkably in wt- and
F137V-PcPAL, mostly due to the changes in dihedral angle
Table 4 Calculated relative binding energies (bRΔE), rounded values of molar fractions of the various covalent N-MIO complexes (x), energy-based
descriptors of the acidity of their pro-S β-hydrogen (cBRΔE) and steric strain of activation (StrainRΔE) for intermediates of compound 1a-d in PcPAL variants. In
the subscript L and D correspond to the actual enantiomer; psc, pst, and u correspond to the s-cis, s-trans product forming, and the unproductive 1a-d–
N-MIO states.
b
c
c
C
c
b
b
b
b
b
PcPAL
Substrate
bRΔE
xL-psc
(%)
xL-pst
(%)
xD
xL-u
StrainRΔEL-psc
StrainRΔEL-pst
cBRΔE
cBRΔE
cBRΔE
-
cBRΔE
L-psc
L-psc
L-pst
L-pst L-psc
(kcal mol-1)
(%)
0.1
0.0 97.9
0.7 57.8
0.0
0.0
0.0
0.8
(%)
0.0
(kcal mol-1)
11.0
22.5
12.9
33.9
-0.7
(kcal mol-1)
16.2
28.5
20.4
42.4
1.1
(kcal mol-1)
(kcal mol-1)
(kcal mol-1)
6.1
1a
1b
1ca
1d
1a
1b
1ca
1da
13.0
22.5
13.6
33.7
-3.3
-3.2
1.4
22.7
99.7
2.1
0.2
0.0
0.0
0.0
4.8
0.1
5.0
-2.2
-1.5
11.9
-11.4
-4.6
-5.4
11.3
-31.6
3.8
-1.8
8.2
-11.5
-10.7
-7.7
0.5
-20.4
-0.3
-3.6
0.1
-6.2
-2.4
-10.8
11.3
wild-type
41.5
93.4
95.2
99.9
60.9
6.6
0.0
0.0
0.0
1.4
5.3
25.3
5.7
6.8
22.9
F137V
0.3 18.1
0.9 80.7
a In these cases multiple psc and pst conformations were obtained, but only the lowest energy representatives were taken into account. b Quantities are related to
the same property of L-Phe with wt-PcPAL, in the form of RΔE=ΔE- ΔEL-Phe;wt-PcPAL. For the detailed description of the calculation method, see the ESI. For the
reasoning of the necessary relativization, see ref. 30. c Molar fractions were calculated based on Boltzmann probabilities incorporating all substrate conformations.
6 | Org. Biomol. Chem., 2017, 00, 1-9
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Journal Name
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between the phenyl ring and the conjugated double bond of bromo- and rac-4-trifluorostyrylalanines to be extVrieewmAretilcyle pOnolioner
-1
DOI: 10.1039/C7OB00562H
the styryl moiety. The more planar the skeleton was; the substrates or reversible inhibitors (k
≤ 0.0001 s ).
cat-pred
better dispersion of the negative charge could happen due to The F137V mutation calculated no significant improvement
the higher degree of conjugation. This implied that in an only for rac-4-trifluorostyrylalanine (kcat-pred≤ 0.0001 s-1).
efficient enzyme design protocol the substrate affinity and Modeling within this mutant indicated comparably high
electronic effects should be monitored simultaneously.
This exploratory model validates the very concept of our rac-styrylalanine (rac-1a
computations and it can be used as a tool to analyze various catalytic activities were predicted for rac-4-bromo- and rac-4-
factors of importance in catalysis and to provide valuable input cyanostyrylalanines
(kcat-pred= 0.120 s-1 and 0.098 s-1,
activity for rac-4-fluorostyrylalanine (kcat-pred= 0.18 s-1) as for
kcat= 0.28 s-1). Somewhat lower
:
for further studies. Although this model was not expected to respectively) being in the range of the activity of rac-4-
predict the apparent kcat with high accuracy, it enabled us to chlorostyrylalanine (rac-1c: kcat= 0.16 s-1).
predict the order of magnitude of the catalytic activity for
Study for ammonia addition onto styrylacrylates 2a-d with PcPALs
several further congeneric substrates.
With our model, we examined the behavior of further Regrettably, no 1a-d could be detected by HPLC in the
substituted styrylalanines bearing bromo, cyano, fluoro, and attempted ammonia addition reactions of styrylacrylates 2a-d
trifluoromethyl substituents at position
4
(Table 5). with either wt-, F137V-, F137A-, or F137G-PcPAL even after 20
Calculations with wt-PcPAL predicted rac-4-fluorostyrylalanine days (Scheme 1, for details see Section 5.6 of ESI). The possible
and rac-4-cyanostyrylalanine to be poor substrates behaving reasons of these results were analyzed by computer modeling
similarly to rac-styrylalanine, rac-1a. Modeling forecast rac-4- in the cases of wt- and F137V-PcPAL. It was not surprising, that
Table 5 Calculated relative binding energies (bRΔE), rounded values of molar fractions of the various covalent N-MIO complexes (x), energy-based
descriptors of the acidity of their pro-S β-hydrogen (cBRΔE) and steric strain of activation (StrainRΔE) for intermediates of further 4-substituted styrylalanines
in PcPAL variants, and predicted (kcat-pred) values for the corresponding reactions. In the subscript L and D correspond to the actual enantiomer; psc, pst,
and u correspond to the s-cis, s-trans product forming, and the unproductive 4-substituted styrylalanine–N-MIO states.
b
c
c
C
c
b
b
b
b
PcPAL
4-Substituenta
bRΔE
xL-psc
(%)
xL-pst
(%)
xD
xL-u
StrainRΔEL-psc
StrainRΔEL-pst
cBRΔE
cBRΔE
kcat-pred
(s-1)
L-psc
L-psc
L-pst
(kcal mol-1)
(%)
0.0
0.0
0.0
0.0
0.0
0.0 100.0
0.0
0.0
(%)
12.0
99.8
97.9
6.6
(kcal mol-1)
13.5
(kcal mol-1)
20.3
(kcal mol-1)
(kcal mol-1)
-1.5
F-
CF3-
Br-
NC-
F-
CF3-
Br-
13.2
38.7
23.3
33.7
-4.3
15.5
-2.6
8.3
88.0
0.2
2.1
93.4
99.3
0.0
95.9
100.0
0.0
0.0
0.0
0.0
0.7
0.0
0.0
0.0
-0.3
-3.7
-1.7
-11.4
-5.7
-6.8
-5.4
-13.7
0.014
40.1
24.2
33.9
0.6
21.5
2.9
13.1
48.0
32.3
42.4
3.6
15.5
8.2
17.8
-3.8
-2.4
-11.5
-6.9
-8.2
<0.0001
0.0001
0.004
0.180
<0.0001
0.120
wild-type
0.0
F137V
4.1
0.0
-1.6
-15.0
NC-
0.098
a Substituent of the further 4-substituted styrylalanines. b Quantities are related to the same property of L-Phe with wt-PcPAL, in the form of RΔE=ΔE- ΔEL-Phe;wt-PcPAL
.
For the detailed description of the calculation method, see the ESI. For the reasoning of the necessary relativization, see ref. 30. c Molar fractions were calculated
based on Boltzmann probabilities incorporating all substrate conformations.
A
B
Figure 4 Models of possible styrylacrylate 2a-arrangements within amino-enzyme (NH2-N-MIO) forms of PcPAL variants. (A) Hydrogen bonds forming
among the carboxylate moiety, a water molecule and R354, N384 of the bidentate and monodentate salt bridge forming poses of 2a within F137V-
PcPAL. (B) The distances of the N atom of NH2 of the NH2-N-MIO from the C2 atom of the two lowest energy mono- (2amono) or bidentate (2abi)
carboxylate binding poses of 2a within F137V-PcPAL. In both panels, the model depicting 2amono (in cyan) corresponds to a product from the productive
N-MIO intermediate (L-1apsc), the model depicting 2abi (in blue) shows a low energy inactive state.
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ARTICLE
Organic & Biomolecular Chemistry
structure of a TchPAM mutant (PDB ID: 4CQ5).22 In the
structure of this enzyme homologous to PcPAL, a continuous
View Article Online
DOI: 10.1039/C7OB00562H
Table 6 Calculated relative ligand binding energies (bRΔE) and rounded
values of molar fractions of mono- and bidentate carboxylate binding
modes (x) for 2a-d [mono and bi in the subscript relate to the lowest
energy ligand pose that forms a monodentate (putatively active pose)
or bidentate (putatively inactive pose) salt bridge].
electron density between R354 and N384 and the carboxylate
of the ligand was observed in the active site complexed with
(E)-cinnamic acid and a water which was consistent with
multiple ligand poses in similar arrangements to that was
found in our models for 2a in PcPAL.
Modeling results suggest that low binding affinities of 2a-d
(especially to wt-PcPAL) and substrate inhibition by the
inactive substrate poses of 2a-d in both PcPAL variants could
contribute to the failure of the ammonia addition. Modeling,
however, could not represent results which could completely
exclude the possibility of successful ammonia addition,
especially in the cases of 2a,b with F137V-PcPAL. Therefore,
further reasons of the failure of ammonia addition can be
proposed: i) the thermodynamic equilibrium of ammonia
PcPAL
Compound
wt-PcPAL
F137V-PcPAL
a
b
b
a
b
b
bRΔEmono
xmono
(%)
xbi
bRΔEmono
xmono
(%)
xbi
-1
-1
kcal mol
(%)
kcal mol
(%)
2a
2b
2c
2d
17.2
25.8
20.4
41.8
1.0
18.0
0.1
97.5
81.9
75.8
92.6
2.2
-0.2
2.0
15.8
77.9
0.6
71.8
14.4
91.3
93.3
0.0
10.4
6.3
a
Quantities are related to the same property of (E)-cinnamate with wt-
PcPAL, in the form of RΔE=ΔE- ΔE(E)-cin;wt-PcPAL. For the detailed description of
the calculation method, see the ESI. For the reasoning of the necessary
b
relativization, see ref. 30. Molar fractions were calculated based on
Boltzmann probabilities incorporating all ligand poses.
addition can be too low to result in detectable amounts of
1a-d, ii) the low water solubility and rather poor affinity of 2a-
result in non-detectable reaction rates product
L-
the relative binding energies of 2a-d indicated poor binding to
wt-PcPAL which was remarkably improved with F137V-PcPAL
(Table 6, bRΔEmono). Modeling revealed allegedly unproductive
ligand poses for 2a-d with both PcPAL variants in almost all
cases with higher binding affinity as compared to the possibly
reactive arrangements of 2a-d. The case of 2a within F137V-
d
/
concentrations, and iii) probably the enzyme adopts
significantly different conformation in 6M ammonia that
makes the computational results invalid under the conditions
of the ammonia addition.
PcPAL is shown in Figure 4. Panel
A depicts the lower energy
arrangement of the putatively inactive conformation of 2a
with a bidentate salt bridge between R354 and the carboxylate
group (in blue) compared to the putatively catalytically active
conformation forming a monodentate salt bridge (in cyan).
Experimental
The detailed description of the materials, analytical methods,
chemical synthesis of substrates rac-1a-d, 2a-d, curves from
Panel
B of Figure 4 depicts models of the possible
kinetic measurements, HPLC chromatograms used for the
determination of conversion and enantiomeric excess values,
as well as the details of the methods used for molecular
modeling are attached as Electronic Supplementary
Information (ESI).
arrangements of 2a within F137V-PcPAL with an elongated
distance between the N atom of NH2 of the NH2-N-MIO and
the C2 atom in the model for putatively inactive conformation
of 2a (in blue) as compared to the corresponding distance in
the model including 2a in reactive conformation (in cyan)
.
Grand canonical Monte Carlo simulations indicated a water
molecule for both binding poses of 2a forming a hydrogen
bond with the carboxylate group of the ligand, albeit in
remarkably different positions. Irrespectively to the
monodentate or bidentate nature of the carboxylate moiety
regarding to the arginine R354, the three oxygen atoms of the
carboxylate group and the water always formed hydrogen
bonds with residues R354 and N384 (Figure 4, Panel A). These
findings agreed with experimental results found in X-ray
Site-directed mutagenesis
The site-directed mutagenesis was performed following the
protocol described by Naismith and Liu.33
The PCR reaction contained 10 ng of template DNA
(recombinant plasmid encoding PcPAL34), 1 μM solution of
primer pair (Table 7), 200 μM dNTPs and 3 units of Pfu DNA
polymerase, filled up to 50 μL with water. The PCR cycles were
initiated at 95 °C for 5 min, followed by 15 amplification cycles.
Table 7. Primers used for site-directed mutagenesis
Primer
Sequence (5’-3’)
Tmpp (oC)*
Tmno (oC)*
F137V-for
F137V-rev
F137A-for
F137A-rev
F137G-for
F137G-rev
PcPAL-for1
PcPAL-for2
GATTAGAGTCTTGAACGCCGGAATTTTCGGAAACGGATC
GGCGTTCAAGACTCTAATCAATTCCTTCTGCAAGGCTCC
GATTAGAGCCTTGAACGCCGGAATTTTCGGAAACGGA
GCGTTCAAGGCTCTAATCAATTCCTTCTGCAAGGCTCCTC
GATTAGAGGCTTGAACGCCGGAATTTTCGGAAACGGA
GCGTTCAAGCCTCTAATCAATTCCTTCTGCAAGGCTCCTC
CAGAACATTACTCCCTGCTTG
56
56
58
58
58
58
-
64
64
67
67
67
67
-
TTGACGTATCCAGAAACAAGG
-
-
* Tmpp and Tmno represents the melting temperature for the primer-primer overlapping sequence and for the primer sequence matched to the
template
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Each amplification cycle consisted of 95 °C for 1 min, at
temperature of Tmno-5 °C for 1 min and 72 °C for 15 min. The
PCR cycles were finished with an annealing step at Tmpp-5 °C
for 1 min and the final extension step at 72 °C for 30 min. The
PCR products were treated with 5 units of DpnI at 37 °C for 2 h
and then 10 μL of each PCR reactions was analyzed by agarose
gel electrophoresis. An aliquot of 3 μL from the above PCR
product was transformed into 100 μL suspension of E. coli XL1-
Blue competent cells (OD600 2.2) by heat shock. The
transformed cells were spread on a Luria-Bertani (LB) plate
containing carbenicillin (34 µg/mL) and chloramphenicol (50
µg/mL) and incubated at 37 °C for 16 h. Two colonies from
each plate were grown and the plasmid DNA was isolated. To
verify the mutations, 400 ng of plasmid DNA was mixed with
50 pmol of T7-for, T7-rev and PcPAL-for1, PcPAL-for2
sequencing primers (Table 6) in a final volume of 15 μL. DNA
sequencing was carried out by Genomed (Debrecen, Hungary).
View Article Online
DOI: 10.1039/C7OB00562H
Conclusions
In this study, the substrate scope of PcPAL was extended, by a
small library of mutants, towards styrylalanine derivatives, a
useful class of synthetically challenging phenylalanine
analogues.
Although L-styrylalanine, L-1a, was accepted as substrate by
wt-PcPAL in the ammonia elimination reaction, low reaction
rates were observed with the wild-type enzyme. Molecular
modeling suggested that the most serious clash between the
aromatic moiety of L-styrylalanine, L-1a, and the side chain of
residue F137 in the hydrophobic binding pocket can be
reduced by introducing residues smaller than F137 by point
mutations.
Single point mutations of F137 resulted a small mutant library
(F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL
showed significantly increased catalytic efficiency and could
Protein expression
transform
L-styrylalanine,
L-1a, with comparable activity that of
the wt-PcPAL with
applied as an effective biocatalyst in the synthetically valuable
kinetic resolutions by ammonia elimination from racemic
L
-Phe. Furthermore, F137V-PcPAL could be
Expression and purification of the wild-type PcPAL and the
F137V, F137A, F137G mutants were performed using the
protocol described earlier.34 The protein purity and
homogeneity was determined using SDS-PAGE and size-
exclusion chromatography (Fig. S37, Fig. S38).
styrylalanine analogues rac-1a
-d providing access to D-
styrylalanines 1a , with highly improved catalytic efficiency
D-
-d
as compared to the wt-PcPAL in the same reactions.
The reverse ammonia addition reactions onto (E)-
Enzyme activity measurements
styrylacrylates 2a
-
d
L
– which would be useful for the synthesis
1a-d – failed with either the wild-type or
Enzyme assays were carried out in triplicate at 30 °C in 1 mL
quartz cuvette using 0.1 M Tris and 0.12 M NaCl (pH 8.8) as
buffer at constant concentration of wt-PcPAL or F137V-PcPAL
(for details, see the ESI), varying the substrate concentration
from 0.025 and 7.8 mM, until substrate saturation occurred.
The kinetic measurements were based on UV-spectroscopy by
of -styrylalanines
L
-
the mutant enzyme variants (F137V, F137A, F137G),
presumably due to the formation of energetically favorable
unproductive binding states of the styrylacrylates 2a
Molecular modeling – used as an aid to explain the results of
this study – revealed unusually high affinities of the
enantiomers 1a-d towards wt-PcPAL, but proved to be
unproductive. Moreover, an unproductive and two productive
conformations were revealed for the -enantiomers 1a-d
-d.
D-
monitoring the production of the acrylic derivatives 2a
wavelengths where the corresponding amino acids rac-1a
showed no absorption (Table S1). Kinetic constants (KM, vmax
-d at
D-
-d
,
L
L-
.
and kcat) were obtained from Michaelis-Menten curves by non-
linear fitting obtained by MATLAB (see the ESI).
Thus, the proportion of the unproductive complexes acting as
potent reversible inhibitors and compared to the active
arrangements of the
important.
L-enantiomers L-1a-d turned out to be
Preparative scale ammonia elimination reactions from rac-1a-d
The enzymatic reactions were performed in 20 mL of Tris (0.1
M, 0.12 M NaCl, pH 8.8), containing styrylalanines (rac-1a-d, 5
mM) and purified PcPAL (0.5 mg of F137V-PcPAL or 1 mg of wt-
PcPAL). Conversion and the enantiomeric excess of the
By the mutation F137V in PcPAL, the less strained arrangement
of substrates 1a-d within the active site along with the more
L-
pronounced populations of conformations with higher
catalytic activities simultaneously led to the beneficial catalytic
efficiency of this mutant. Modeling the behavior of further 4-
substituted styrylalanines with PcPAL variants predicted the
F137V mutation beneficial for the KRs of 4-fluoro-, 4-cyano-
and 4-bromostyrylalanines, but non-effective for the KR
process of 4-triflouromethylstyrylalanine.
The results of this study opens new perspectives for substrate
scope expansion of PcPAL towards synthetically challenging
phenylalanine analogues with increased length of the carbon
skeleton between the aromatic moiety and the α-stereogenic
carbon.
residual
D-1a-d were monitored using reverse phase high
performance liquid chromatography (HPLC) analysis (see the
ESI). Samples (30 μL) were taken from the reaction mixture
after different time intervals, quenched by adding an equal
volume of MeOH, vortexed and centrifuged (13400 rpm,
12100 g, 2 min). The supernatant was used directly for HPLC
analysis after transferring through a 0.22 μm nylon membrane
filter. In case of enzyme reactions over longer reaction time,
another batch of enzyme was added after each 48 h period.
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Acknowledgements
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DOI: 10.1039/C7OB00562H
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