Tailored Mutants of Phenylalanine Ammonia-Lyase from Petroselinum crispum for the Synthesis of Bulky l- and d-Arylalanines

Article abstract of DOI:10.1002/cctc.201800258

Tailored mutants of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) were created and tested in ammonia elimination from various sterically demanding, non-natural analogues of phenylalanine and in ammonia addition reactions into the corresponding (E)-arylacrylates. The wild-type PcPAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PcPAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates. However, combined mutations that involve I460 besides the well-studied F137 led to mutants that exhibited activity in ammonia addition as well. The synergistic multiple mutations resulted in substantial substrate scope extension of PcPAL and opened up new biocatalytic routes for the synthesis of both enantiomers of valuable phenylalanine analogues, such as (4-methoxyphenyl)-, (napthalen-2-yl)-, ([1,1′-biphenyl]-4-yl)-, (4′-fluoro-[1,1′-biphenyl]-4-yl)-, and (5-phenylthiophene-2-yl)alanines.

Full text of DOI:10.1002/cctc.201800258

Accepted Article
Title: Tailored mutants of phenylalanine ammonia-lyase from
Petroselinum crispum for the synthesis of bulky L- and D-
arylalanines
Authors: Alina Filip, Emma Zsofia Aletta Nagy, Diana Tork Souad,
Gergely Bánóczi, Monica Ioana Toşa, Florin Dan Irimie,
Laszlo Poppe, Csaba Paizs, and Laszlo Csaba Bencze
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ChemCatChem
10.1002/cctc.201800258
FULL PAPER
Tailored mutants of phenylalanine ammonia-lyase from
Petroselinum crispum for the synthesis of bulky L- and D-
arylalanines
Alina Filip,#[a] Emma Z. A. Nagy,#[a] Souad D. Tork, Gergely Bánóczi, Monica I. Toșa, Florin D.
[a]
[a]
[a]
Irimie, [a] László Poppe,*[a],[b] Csaba Paizs,*[a] and László C. Bencze*[a]
[
5-7]
Abstract: Tailored mutants of phenylalanine ammonia-lyase from
Petroselinum crispum (PcPAL) were created and tested in ammonia
elimination from various sterically demanding, non-natural analogues
of phenylalanine and in ammonia addition reactions onto the
corresponding (E)-arylacrylates. The wild-type PcPAL was inert or
exhibited quite poor conversion in both reactions with all members of
the substrate panel. Proper single mutations of residue F137 and the
highly conserved residue I460 resulted in PcPAL variants being
active in ammonia elimination but having still poor activity in
ammonia additions onto bulky substrates. However, combined
mutations involving I460 besides the well-studied F137 led to
mutants exhibiting activity in ammonia addition as well. The
synergistic multiple mutations resulted in substantial substrate scope
extension of PcPAL and opened up novel biocatalytic routes for the
synthesis of both enantiomers of valuable phenylalanine analogues,
such as (4-methoxyphenyl)-, (napthalen-2-yl)-, ([1,1'-biphenyl]-4-yl)-,
4.3.1.24/25)
from
Petroselinum
crispum
(PcPAL),
[
8-10]
Rhodotorula glutinis or graminis (RgPAL or RgrPAL),
and
Anabaena variabilis (AvPAL)[11,12] were used most of the time as
biocatalysts in whole cells or as purified enzymes for ammonia
elimination or for the reverse ammonia addition yielding a wide
range of L- and D-arylalanines.
Although wild-type PALs exhibited broad substrate tolerance
towards various cinnamic acid derivatives in the ammonia
addition, synthetically valuable 4-substituted cinnamic acids with
or electron-
)[9,15,16] substituents proved to
be poor substrates. Protein engineering strategies focused
mainly on residue F137 in the hydrophobic binding pocket of
or on the sterically analogous F107 and H143 of
AvPAL[14] and RgrPAL,[10] respectively. These mutations
enhanced the catalytic properties of PALs in ammonia addition
electron-withdrawing but bulky (4-NO
donating (4-CH , 4-OCH , 4-NH
2
, 4-Ph)[
13,14]
3
3
2
PcPAL[
13,17]
[
[
3,[
4]
(4'-fluoro-[1,1'-biphenyl]-4-yl)-, and (5-phenylthiophene-2-yl)alanines.
2
onto 4-NO , 4-Br, and 4-F-substituted cinnamic acids 14] 13]
and in the ammonia elimination of styrylalanines[[17]7], however,
none of the PAL mutants showed activity in the ammonia
addition onto (4-phenyl)cinnamic acid[14]
or styrylacrylic
Introduction
acids[[17]7]. Screening novel PALs from various organisms
revealed variants which favored the acceptance of substituted
cinnamic acids with electron-donating substituents, but
conversion of valuable 4-methoxyphenylacrylate still remained a
challenge.[16]
The synthesis of natural and unnatural aromatic amino acids in
homochiral form is an important challenge of preparative
chemistry, highlighted by the significant interest towards these
building blocks in the development of therapeutic peptides and
proteins.[1-3] An attractive enzymatic route to enantiomerically
pure α- or β-aromatic amino acids involves the use of aromatic
ammonia-lyases (ALs) and 2,3-aminomutases (AMs)[4] acting by
the aid of an autocatalytically-formed 3,5-dihydro-5-methylidene-
Herein we report
a thorough mutational analysis of the
hydrophobic binding pocket of PcPAL with the aim to develop
PAL biocatalysts for the production of bulky and valuable L- and
D-arylalanines. These compounds offer new possibilities to
extend the chemical space available to biomedicine (peptides,
proteins, peptidomimetics) and chiral small molecule drugs. The
envisaged ([1,1'-biphenyl]-4-yl)alanines are constituents of
4H-imidazol-4-one (MIO) electrophilic prosthetic group. Among
the so-called MIO-enzymes, phenylalanine ammonia-lyases (EC
various bioactive peptides[ , such as peptidase, protease or
18]
[14]
cancer-related histone lysine demethylase KDM4A[ inhibitors.
19]
[
a]
Ms. A. Filip, Ms E.Z.A. Nagy, Ms D. T. Souad, Dr. G. Bánóczi, Prof.
M.I. Toşa, Prof. F.D. Irimie, Prof. L. Poppe, Prof. C. Paizs, Dr. L.C.
Bencze
(
Naphthalen-2-yl)alanine is frequently used as a phenylalanine
analogue in the development of peptides,[20,21] while (4-
methoxyphenyl)alanine is a chiral intermediate in the synthesis
of the antihypertensive drug tamsulosin.[ (5-Phenylthiophene-
Biocatalysis and Biotransformation Research Centre
Faculty of Chemistry and Chemical Engineering
Babeș-Bolyai University of Cluj-Napoca
22]
2-yl)alanine and its novel derivatives represent also attractive
Arany János Str. 11, RO-400028 Cluj-Napoca, Romania
E-mail: cslbencze@chem.ubbcluj.ro, paizs@chem.ubbcluj.ro
Prof. L. Poppe
phenylalanine analogue targets due to the heteroaryl motif
featured in endothelin convertase[23] and Factor IX/XI
[
b]
24]
Department of Organic Chemistry and Technology
Budapest University of Technology and Economics
Műegyetem rkp. 3, H-1111 Budapest, Hungary
inhibitors.[ Amide derivatives of styrylalanine were identified as
potent peptidyl-prolyl isomerase (PPIase) inhibitors at Pfizer.[25]
The origin of potency was attributed to the (E)-ethene-1,2-diyl
linker increasing the distance between the aromatic moiety and
the chiral alanine moiety.
#
These authors contributed equally to the work.
Supporting information for this article is given via a link at the end of
the document
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It is worth noting that some of the target compounds (Scheme 1)
had already been tested before and exhibited little or no
conversions or even inhibitory activity with wild-type
PALs.[9,14,15,17] Moreover, no mutant PAL variants were known to
possess activity in the corresponding ammonia elimination and
ammonia addition reactions, except for the deamination of
styrylalanines.[ The initial tests of this study also confirmed the
insufficient catalytic activity of wt-PcPAL upon the substrate
panel.
(i.e. V, A) providing a limited number of single or multiple residue
mutants of PcPAL (Table S1).
To exclude the influence of mutation-induced improper folding
on enzyme activity, the oligomerization state and thermal
unfolding of the isolated and purified mutants were compared
with those of the wt-PcPAL. Retention volumes from size-
exclusion chromatography (ESI, figure S2) revealed that all
PcPAL variants were properly folded and existed in the native,
tetrameric form, similarly to the wild-type enzyme. The slight
17]
m
variations in thermal unfolding temperatures (T ), determined by
differential scanning fluorimetry measurements, indicated that
the mutations did not affect protein folding (see Table S3 in ESI).
The only exception was the I460A mutation which decreased the
m
T value significantly (from 75±1.5 °C to 51±1.2 °C, see Table
S4, Figure S5 in ESI) without affecting the tetrameric fold
detected by size exclusion chromatography. Since later tests
showed that I460A-PcPAL was catalytically active (Tables S2-
S19 in ESI), we presumed that the mutation corrupted only the
thermal stability without disrupting the main folding patterns
related to enzyme activity.
Figure 1. Active site model of PcPAL with (E)-cinnamic acid as a modeled
ligand and the surrounding residues within less than 5 Å distance. Colors of
amino acid side chains refer to their position compared to the plane of
substrate: black – within, red – above, blue – below the plane. Hydrophobic
binding pocket residues in boxes were exchanged individually or in
combination to smaller hydrophobic amino acids V or A.
Scheme 1. The ammonia elimination (A) and ammonia addition (B) reactions
tested by the PcPAL variants.
Results and Discussion
CASTing[26] or directed evolution methods requiring high-
Next, the generated single mutant PcPAL library (Table S1) was
tested with the targeted substrate panel in ammonia elimination
from arylalanines rac-1a-i (Scheme 1A) and in ammonia addition
onto arylacrylates 2a-i (Scheme 1B). Results from the ammonia
eliminations revealed that besides the known F137 to V or A
mutations,[13,[17]7] mutation of another highly conserved residue
I460 (see Figure S1 in ESI) to valine or alanine increased the
activity significantly towards almost all substrates compared to
the wild-type enzyme (Table 1 and Tables S5-13 in ESI).
throughput enzyme assays (HTS) of large mutant libraries to
generate mutants of PcPAL accepting the bulky target
compounds as substrates were avoided, due to cell membrane
penetration issues of the large hydrophobic substrates with
whole cell PAL-biocatalysts. Instead of such HTS-based
methods, the structure-driven approach was selected and based
[
[
4,[
7]
on steric clash reduction concepts. 14] 17] , residues L134,
F137, L138, L206, L256, and I460 from the hydrophobic binding
site of PcPAL (Figure 1) were exchanged to smaller amino acids
Whilst wt-PcPAL could convert the members of the tested
substrate panel quite poorly (crac-1a= 3%; crac-1b= 6%; crac-1c-i
<
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FULL PAPER
i (Table 1). The lowest enhancement was achieved from rac-1i,
a
(
structural analogue of the known wt-PcPAL-inhibitors
benzo[b]furan-3-yl)- and (benzo[b]thiophene-3-yl)alanines[
6]
Table 1. Activity of wild-type (wt) PcPAL compared to the best PcPAL
single mutants in the ammonia elimination reaction of rac-1a-i
where only mutant F137A provided the arylacrylate 2i, but
regrettably at low conversion (crac-1i= 6%).
Substrate
rac-1a
rac-1a
rac-1a
rac-1b
rac-1b
rac-1b
rac-1c
rac-1c
rac-1c
rac-1d
rac-1d
rac-1d
rac-1e
rac-1e
rac-1e
rac-1f
R group
PcPAL variant[a]
wt
c[b]
3
Despite the flourishing extension of substrate scope of wt-
PcPAL in ammonia elimination, in the reverse ammonia addition
reaction the single mutants of PcPAL showed improved activity
only with 4-methoxyphenyl (2a)-and napthalen-2-ylacrylic acid
4-methoxy
4-methoxy
F137
I460V
wt
37
39
6
(
2b), while for more bulky substrates 2d-h and styrylacrylate (2c)
4-methoxy
no conversions could be detected (Tables S14-22 in ESI).
To explore the advantageous synthetic potential of ammonia
addition onto arylacrylates (100% theoretical yield; use of
synthetically accessible, achiral starting materials), further
mutations of neighboring residues were introduced to the single
mutants with the best activities. In this way, a focused library of
double and triple mutants involving I460, F137 and L138 (Table
S1 in ESI) was obtained and tested in both kinds of PAL
mediated reactions (Tables S5-22 in ESI). Analogously to the
single mutant PcPALs, the I460A mutation altered the thermal
unfolding profile of double and triple mutants as well (Figure S5),
but the detected catalytic activities and the native tetrameric fold
indicated that overall folding was not seriously altered even in
case of double and triple mutants.
napthalen-2-yl
napthalen-2-yl
I460V
F137V
wt
37
39
<1
~50
~50
<1
8
napthalen-2-yl
styryl
styryl
I460V
F137V
wt
styryl
biphenyl-4-yl
biphenyl-4-yl
I460V
F137A
wt
Multiple mutations of PcPAL could result in
enhancement of conversion in ammonia elimination from (4’-
fluoro-[1,1’-biphenyl]-4-yl)alanine (rac-1e: 39% with
a moderate
biphenyl-4-yl
35
<1
37
39
<1
35
44
<1
19
~50
<1
10
~50
<1
6
4’-fluorobiphenyl-4-yl
4’-fluorobiphenyl-4-yl
4’-fluorobiphenyl-4-yl
5-phenylthiophen-2-yl
5-phenylthiophen-2-yl
5-phenylthiophen-2-yl
2’-chloro-5-phenylthiophen-2-yl
2’-chloro-5-phenylthiophen-2-yl
2’-chloro-5-phenylthiophen-2-yl
4’-chloro-5-phenylthiophen-2-yl
4’-chloro-5-phenylthiophen-2-yl
4’-chloro-5-phenylthiophen-2-yl
2-phenylthiazol-4-yl
2-phenylthiazol-4-yl
F137A/I460V-PcPAL, 18% with F137A/L138V-PcPAL vs. 15%
with F137A-PcPAL; see Table S9 in ESI) and for (5-
phenylthiophen-2-yl)alanine (rac-1f: 48% with F137A/L138V-
PcPAL vs. 44% with F137A-PcPAL; see Table S10 in ESI).
Similarly, in case of ammonia additions onto 4-methoxyphenyl
F137V
F137A
wt
(
2a)- and napthalen-2-ylacrylic acid (2b) no significant increase
rac-1f
F137V
F137A
wt
in conversion values was provided by mupltiple mutations (Table
S14, S15).
rac-1f
However, PcPALs having simultaneous mutations of F137 and
I460 gave promising results in ammonia additions onto 2c,d. In
these cases no product could be detected with wt-PcPAL or
either single mutants of PcPAL, but 22%, respectively 27%
conversion could be achieved with the F137(V,A)/I460V double
mutants (Table 2; Tables S16,S17 in ESI). Arylacrylates 2e-h
proved to be moderate substrates even for the F137A/I460V
double mutant (conversions between 3-8% after 20 h, Tables
S18-21 in ESI), while no conversion of (2-phenylthiazol-4-
yl)acrylic acid (2i) could be achieved with the investigated
multiple mutant PcPALs (Table S22 in ESI).
rac-1g
rac-1g
rac-1g
rac-1h
rac-1h
rac-1h
rac-1i
F137V
F137A
wt
I460V
F137A
wt
Our mutational analysis revealed that in most cases single
mutations of F137 and I460 in PcPAL were sufficient to perform
ammonia elimination from bulky amino acids decently and
additional mutations did not significantly improve conversions.
However, double mutants of PcPAL involving F137, I460 were
required to achieve adequate ammonia addition activity with
bulky arylacrylates 2c-h. In case of less bulky substrates 2a,b
active single mutants of PcPAL could be identified as well.
These data clearly demonstrated that multiple mutations
rac-1i
F137A
[
a] PcPAL variant: 50 µg, reaction volume: 500 µL, medium: Tris-buffer
(100 mM Tris.HCl, pH 8.8, 20 mM β-cyclodextrin), substrate
concentration: 1 mM; assays were performed in 1.5 mL glass vials sealed
with PTFE septum at 30 °C, 200 rpm for 16 h;
[
b] conversion values (%)
[
27]
exhibited a strong, non-additive, cooperative effect on PcPAL
1%, even after longer reaction times up to 48 h), mutants I460V
activity in the ammonia addition reaction.
and F137V/A provided medium to high conversions from rac-1a-
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The fact that individual mutations of L134, L206, L256, and L138
L138 as well as the double and triple mutants of L138 with the
neighbor, activity modulator residues F137 and I460 didn’t
provide any increase in conversion of the tested substrate panel
in neither reaction direction highlighted the importance of
residue I460, besides the well-studied residue F137, and their
combined mutations for the substrate specificity modulation of
PcPAL, especially in the synthetically valuable ammonia addition
reaction.
compared to purified enzymes. In spite of our all efforts (using
living or lyophilized whole cells, various biocatalyst-substrate
ratio or temperature), results of whole cell biotransformations
with these bulky and hydrophobic substrates were irreproducible
even within the same batch of cells suggesting cell
internalization difficulties of the bulky hydrophobic substrates.
The reproducibility of experiments with cell lysates supported
this hypothesis, but provided poor quality analytical data
(appearance of additional signals in HPLC chromatograms).
Since different batches of purified enzymes exhibited negligible
biocatalytic variability and clean analytical data, all further
experiments were performed with isolated PcPALs.
Table 2. Activity of the wild-type (wt) PcPAL compared to the best PcPAL
mutants in the ammonia addition reaction of 2a-h
The low solubility (<1 mM) of substrates 1b,d-i, in the reaction
buffer of ammonia eliminations was addressed already during
the initial screening tests. Although by using DMSO or MeOH as
cosolvents (5,10,20 v/v%) the solubility of rac-1d could be
increased to 2-3 mM, conversions dropped from 37% after 16 h
to 16% at 10% cosolvent level and down to zero at 20%
cosolvent level. Finally, solubilization of rac-1d up to 2.5 mM
concentration was achieved by forming inclusion complex with
5-20 mM β-cyclodextrin[28] without altering the enzyme activity.
Thus, activity screens in ammonia elimination with rac-1a-i were
performed in the presence of 20 mM β-cyclodextrin (Table 1).
Unfortunately, the low substrate solubility prevented us from the
determination of Michaelis-Menten curves approaching
substrate saturation. Despite the apparent solubility increase by
β-cyclodextrin, the unknown actual concentrations of non-
complexed substrate and product hindered the determination of
kinetic constants.
Substrate
R group
PcPAL variant[a]
c[b]
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
a
a
b
b
c
c
d
d
e
e
f
4-methoxy
wt
<1
32
<1
55
<1
22
<1
27
<1
8
4-methoxy
F137V/I460V
napthalen-2-yl
wt
napthalen-2-yl
F137V
styryl
wt
styryl
F137V/I460V
biphenyl-4-yl
wt
biphenyl-4-yl
F137A/I460V
4’-fluorobiphenyl-4-yl
4’-fluorobiphenyl-4-yl
5-phenylthiophen-2-yl
5-phenylthiophen-2-yl
2’-chloro-5-phenylthiophen-2-yl
2’-chloro-5-phenylthiophen-2-yl
4’-chloro-5-phenylthiophen-2-yl
4’-chloro-5-phenylthiophen-2-yl
wt
The solubility of acrylic derivatives 2a-i in the high concentration
ammonia buffer was higher without any additive (2.5 mM, Table
F137A/I460V
wt
2), but still not enough to obtain full Michaelis-Menten curves.
<1
6
Next, the influence of various ammonium sources was tested
(2,4,6 M ammonia or ammonium carbamate) on the PcPAL-
catalyzed ammonia addition of (napthalen-2-yl)acrylic acid (2b).
The best results in terms of conversion values and enantiotopic
selectivities were achieved using 4M ammonium carbamate
f
F137A/I460V
wt
g
g
h
h
<1
3
F137A/I460V
wt
(
Table S23 in ESI), in accordance with the optimal conditions
<1
2
reported for the PAL-catalyzed ammonia addition onto 3-
fluorocinnamic acid.[[12]]
Finally, the PcPAL-catalyzed reactions of the entire substrate
panel (rac-1a-i, 2a-i) were performed under the optimal reaction
conditions, monitoring the conversions and enantiomeric excess
values of D- and L-1a-i.
F137A/I460V
[a] PcPAL variant: 50 µg, reaction volume: 500 µL, medium: 6M NH
3
buffer
(pH 10, adjusted with CO ), substrate concentration: 1 mM; assays were
2
performed in 1.5 mL glass vials sealed with PTFE septum at 30 °C, 200
rpm for 20 h.
The maximal conversions in the kinetic resolutions (KRs) as well
as the maximal ee of the unreacted D-enantiomer (D-1a-h) have
been reached in all but one case in relatively short reaction
times (14-40 h, Table 3). In ammonia elimination from (2-
phenylthiazol-4-yl)alanine (rac-1i) the reaction stopped at low
conversion (10%) suggesting product inhibition. This hypothesis
was confirmed by the inhibitory effect of 2i upon the wt-PcPAL
catalyzed ammonia elimination from L-Phe (see ESI).
Furthermore, ammonia additions onto 2i with wt- or mutant
PcPALs did not succeed suggesting that structural analogues of
(benzo[b]furan-3-yl)- and (benzo[b]thiophene-3-yl)acrylates,
known wt-PAL-inhibitors[ still behave as competitive inhibitors
also for mutant PcPALs.
[
b] conversion values (%)
With the most active mutants in hands (Tables 1,2), the reaction
conditions in terms of activity and selectivity were optimized
using ([1,1’-biphenyl]-4-yl)alanine (rac-1d) and (napthalen-2-
yl)acrylic acid (2b) as models for the ammonia elimination and
addition, respectively.
Reactions of rac-1d and 2b were investigated also using whole
cells of E. coli expressing the corresponding PcPAL mutants to
take advantage of the possible lower production costs and
increased stability, characteristic for whole cell PAL biocatalysts
[6]]
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The ammonia addition reactions onto 2a-h proceeded somewhat
slower (Table 4) and, after longer incubation times (>20-24 h),
deactivation of PcPALs was observed due to the harsh reaction
conditions. This issue was solved by supplementing the
reactions with fresh batches of enzyme after each 24 h of
incubation. In almost all cases 80-85% of the final conversions
could be reached within the first 28-36 h, followed by slow
increase of conversions until 70 h reaction time (see Figure S7-
S12, progression curves of ammonia addition reaction) .The fact
that after additional 48 h no further reaction progress could be
detected indicated that an equilibrium state was reached.
and 73%) and excellent enantiomeric excess (eeL-1a and eeL-1b
>99%), while conversion of styrylacrylate (2c) by F137V/I460V-
PcPAL remained low (23%) but selective (eeL-1c >99%). (1,1’-
Biphenyl)acrylates 2d,e were transformed with good to
moderate conversions (68% and 50%) but with non-complete
enantiotopic selectivity (eeL-1d= 82%, eeL-1e= 95%; see Table 4).
4
The fact that NaBH -reduced PcPALs or the MIO-less
S203A/F137A/I460V-PcPAL variant proved to be inactive in
these reactions ruled out the occurrence of the competing, D-
selective MIO-less reaction route.[29] The (5-phenylthiophen-2-
yl)acrylates 2f-h were transformed much slower, reaching
conversions of only 3-9% within 70 h.
The enhanced conversions of L-1a-h with the proper mutants
can be rationalized by the better substrate affinities and also by
the higher turnover numbers due to better stabilization of the
reaction transition states, in accordance with our previous
computer-aided results.[17] Modeling of the N-MIO covalent
enzyme-substrate complexes, obtained with induced-fit covalent
docking, confirmed that substrate affinities increased
dramatically with the most active mutants over wt-PcPAL in
almost all the cases (Table 5).
Table 3. Yield and enantiomeric excess of D-1a-h after the PcPAL-
catalyzed ammonia eliminations from rac-1a-h at ~50% conversion.
cc
PcPAL variant
I460V
t
react (h)
Y [a] (%)
45
eeD-1a-h (%)
>99
rac-1a
rac-1b
rac-1c
rac-1d
rac-1e
rac-1f
rac-1g
rac-1h
16
17
14
40
40
16
16
16
F137V
46
>99
F137V
43
>99
F137A
47
>99
Table 5. Calculated relative binding energies (
b
RΔE) of L-1a-h in wt-PcPAL
F137A/I460V
F137A/L138V
F137A
45
>99
and in the most active PcPAL variants. Subscripts WT and MA correspond to
wild-type and most active mutant, respectively. Worth to note that these
quantities are not meant to computationally determine actual binding
energies but to approximate them only.
46
>99
42
>99
R
Substrate
Most active
PcPAL mutant
b
RΔEWTa
(kcal mol )
b
RΔEMAa
(kcal mol )
b
RΔEMA
-
b
ΔEWT
F137A
39
>99
-1
-1
-1 a
(kcal mol )
[
a] the reaction yields were determined from the preparative scale
L-1a
L-1b
L-1c
L-1d
L-1e
L-1f
I460V
F137V
2.9
3.7
-11.2
-4.0
-3.5
12.6
-5.3
0.3
0.8
ammonia eliminations (for reaction conditions see ESI, chapter 6.6.)
4.9
-16.1
-17.7
-33.3
-22.9
-25.0
-31.8
-33.1
F137V
13.7
29.8
35.5
19.7
32.1
40.6
Table 4. Conversion of PcPAL-catalyzed ammonia additions onto 2a-h and
yield and enantiomeric excess of the products L-1a-h after 70 h reaction
time.
F137A
F137A/I460V
F137A/L138V
F137A
Substrate
PcPAL variant
F137V/I460V
F137V
c (%)
74
73
23
68
50
6
Y[a] (%)
65
eeL-1a-h (%)
>99
>99
>99
82
2
2
2
2
2
2
2
2
a
b
c
d
e
f
L-1g
L-1h
61
F137A
7.5
F137V/I460V
F137A/I460V
F137A/I460V
F137A
19
[
a] Binding energies are related to the same property of L-Phe with wt-
59
R
PcPAL, in the form of
of the method of calculation and reasoning of the necessary relativization,
see Supporting Information.
b
ΔE=
b
ΔE- ΔEL-Phe;wt-PcPAL. For the detailed description
b
43
95
n.d
n.d
n.d
n.d
g
h
F137A/I460V
F137A/I460V
9
n.d
Figure 2 illustrates the reason for affinity increase of L-1e where
F137A/I460V-PcPAL provides a larger active site volume and
much less area of close contact of the N-MIO covalent enzyme-
substrate complex with sites A137 and I460 (Figure 2C) and a
significant shift of the ligand position (Figure 2B) as compared to
wt-PcPAL (Figure 2A). Comparisons of the N-MIO intermediates
from the other L-arylalanines L-1b-d,f-h, indicated similar
3
n.d
[a] the reaction yields were determined from the preparative scale
ammonia additions (for reaction conditions see ESI, chapter 6.7)
Accordingly, 4-methoxycinnamic acid (2a) and (napthalen-2-
yl)acrylic acid (2b) were transformed with good conversion (74%
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10.1002/cctc.201800258
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situations (see ESI). The only exception was L-1a, with a nearly
unaffected affinity, which in turn suggested that the catalytic
enhancement stemmed solely from the other presumed factor, a
higher turnover number.
reaction the high energy intermediate and thus the transition
state (TS) resembles better to the substrate state, while the TS
of an endothermic reaction resembles better to the products
state. In our case, the reverse ammonia addition reaction is
known to be endothermic, therefore the N-MIO intermediate
structure is not appropriate to draw conclusions on the affinity
situations for the arylacrylates. Moreover, the enzyme most
probably adopts a different conformation under high ammonia
concentrations invalidating our computational results for
ammonia addition. This was supported by analysis of the
thermal unfolding profiles of wt-PcPAL (Figure S6) and
F137A/I460V-PcPAL (Figure 3) at different ammonia
concentrations indicating shifts of melting temperature (T
m
) by
10-12 °C at the highest ammonia concentration as compared to
that determined in Tris buffer.
A)
B)
C)
m
Figure 3. The thermal unfolding temperature (T ) of F137A/I460V-PcPAL in
media with different ammonia content (20 mM Tris.HCl, pH 9; and 1 M, 2 M, 4
M and 6 M NH -buffer, with pH 9.5 adjusted by CO ) determined by nanoDSF
Prometheus NT.48). Fluorescence intensity ratios F350/F330 and their first
3
2
(
derivative are represented as a function of the applied linear thermal ramp.
Finally, synthetic applicability of the tailored PcPAL mutants was
demonstrated by performing KRs from racemic arylalanines rac-
1a-h by ammonia elimination digesting the L-enantiomers
(
Scheme 1A) and by the enantiotopic selective ammonia
addition reactions onto 2a-e (Scheme 2B) at larger scale (0.25
mmol substrate, for details, see ESI). The preparative scale
reactions of 2f-h were omitted due to the quite low equilibrium
conversions. During the preparative scale reactions no
significant alterations of conversions, reaction times, and
enantiomeric excess values have been observed as compared
to the analytical scale bioconversions. The corresponding
unreacted D-enantiomers D-arylalanines D-1a-h (Table 3) and
the produced L-arylalanines L-1a-e (Table 4) were isolated
conveniently by Dowex cation-exchange chromatography in
good to moderate yields.
Figure 2. Catalytically active N-MIO intermediate models of L-1e within wt-
PcPAL (panel A) and F137A/I460V-PcPAL (panel C). Volumes inside the
mesh represent the cavity provided by the active site of the corresponding
enzyme. Panel B represents the combination of panel A and C depicted with
the mesh for wt-PcPAL only. Mutational sites and ligand pose are colored
magenta in wt-PcPAL and blue in F137A/I460V-PcPAL. Coloring of the mesh
represents close contacts with the corresponding residues of the mutational
site.
Worth to note that prior to this work, the stereoconstructive
ammonia addition onto the bulky arylacrylates 2a-h were
unprecedented and no data were reported on PAL mediated
routes to amino acids L-1a-e. Similarly, no reports existed on
successful PAL mediated ammonia elimination reactions from
Computational results, however, proved to be inconsistent with
the experimental results of ammonia additions. Apart from
potential parameterization problems of atomic interactions in our
model, two reasons can rationalize this observation. One reason
is the Hammond’s postulate which states that in an exothermic
(
[1,1’-biphenyl]-4-yl)-, (napthalen-2-yl)-, (4-methoxyphenyl)- and
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10.1002/cctc.201800258
FULL PAPER
(
5-phenylthiophen-2-yl)alanines
(1a,b,d-h)
yielding
the Acknowledgements
corresponding amino acids D-1a,b,d-h.
The fact that PALs are known to present difficulties in
transforming substrates with electron-donating ring substituents
and the recent efforts focusing on the discovery of novel PALs
with such activity[16] highlights the excellent results obtained for
the synthesis of both L- and D-4-methoxy-phenylalanine (L- and
D-1a).
Financial support for project PROMYS, (Grant Nr.
IZ11Z0_166543) from the Swiss National Science Foundation
(
1
SNSF) and for project NEMSyB, ID P37_273, Cod MySMIS
03413 [funded by National Authority for Scientific Research
and Innovation (ANCSI) and European Regional Development
Fund, Competitiveness Operational Program 2014-2020 (POC),
Priority axis 1, Action 1.1] is gratefully acknowledged.
Pharmaceutically important ([1,1’-biphenyl]-4-yl)alanines were
subjects of recent AvPAL mediated biotransformations where
the studied AvPAL variants showed no activity in ammonia
addition onto 4-phenylcinnamic acid, thus a chemoenzymatic
procedure was required involving the AvPAL mediated synthesis
of L-(4-bromophenyl)alanine followed by Pd-catalyzed Suzuki-
coupling.[ In this frame, the tailored multiple mutant PcPAL-
based processes reported here represent the first direct
enzymatic route towards both enantiomers of ([1,1’-biphenyl]-4-
yl)alanines L- and D-1d,e.
Conflict of interest
The authors declare no conflict of interest.
14]
Keywords: biocatalysis • phenylalanine ammonia-lyase • non-
natural amino acids • protein engineering • substrate scope
extension
As future perspectives, combination of tailored PcPAL mutants
with the recently reported immobilization techniques can lead to
their use in continuous-flow reactors,[30-32] providing accessibility
for the industrial synthesis of sterically demanding non-natural
arylalanines.
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Entry for the Table of Contents
FULL PAPER
Alone is not enough. Tailored
mutants of phenylalanine ammonia-
lyase from Petroselinum crispum
Alina Filip, Emma Z. A. Nagy, Souad D.
Tork, Gergely Bánóczi, Monica I. Toșa,
Florin D. Irimie, László Poppe,* Csaba
Paizs,* and László C. Bencze*
(
PcPAL) were created and tested in
ammonia elimination reactions of
sterically demanding, non-natural
analogues of phenylalanine and in
ammonia addition reactions onto the
corresponding (E)-arylacrylates. The
synergistic multiple mutations resulted
in substantial substrate scope
extension of PcPAL.
1 – 8
Tailored mutants of phenylalanine
ammonia-lyase from Petroselinum
crispum for the synthesis of bulky
L- and D-arylalanines
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