Influence of the aromatic moiety in α- And β-arylalanines on their biotransformation with phenylalanine 2,3-aminomutase from: Pantoea agglomerans

Article abstract of DOI:10.1039/c6ra02964g

In this study enantiomer selective isomerization of various racemic α- and β-arylalanines catalysed by phenylalanine 2,3-aminomutase from Pantoea agglomerans (PaPAM) was investigated. Both α- and β-arylalanines were accepted as substrates when the aryl moiety was relatively small, like phenyl, 2-, 3-, 4-fluorophenyl or thiophen-2-yl. While 2-substituted α-phenylalanines bearing bulky electron withdrawing substituents did not react, the corresponding substituted β-aryl analogues were converted rapidly. Conversion of 3- and 4-substituted α-arylalanines happened smoothly, while conversion of the corresponding β-arylalanines was poor or non-existent. In the range of pH 7-9 there was no significant influence on the conversion of racemic α- or β-(thiophen-2-yl)alanines, whereas increasing the concentration of ammonia (ammonium carbonate from 50 to 1000 mM) inhibited the isomerization progressively and decreased the amount of the by-product (i.e. (E)-3-(thiophen-2-yl)acrylic acid was detected). In all cases, the high ee values of the products indicated excellent enantiomer selectivity and stereospecificity of the isomerization except for (S)-2-nitro-α-phenylalanine (ee 92%) from the β-isomer. Substituent effects were rationalized by computational modelling revealing that one of the main factors controlling biocatalytic activity was the energy difference between the covalent regioisomeric enzyme-substrate complexes.

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Influence of the aromatic moiety in α- and β-arylalanines on their
biotransformation with phenylalanine 2,3-aminomutase from
Pantoea agglomerans†
Received 00th January 20xx,
Accepted 00th January 20xx
A. Vargaa, G. Bánóczib, B. Nagya, L. C. Benczea, M. I. Toșaa, Á. Gellértc, F. D. Irimiea, J. Réteyd,
L. Poppeb*,e and C. Paizsa*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this study enantiomer selective isomerization of various racemic α- and β-arylalanines catalysed by phenylalanine
2,3-aminomutase from Pantoea agglomerans (PaPAM) was investigated. Both α- and β-arylalanines were accepted as
substrates when the aryl moiety was relatively small, like phenyl, 2-, 3-, 4-fluorophenyl or thiophen-2-yl. While
2-substituted α-phenylalanines bearing bulky electron withdrawing substituents did not react, the corresponding
substituted β-aryl analogues were converted rapidly. Conversion of 3- and 4-substituted α-arylalanines happened
smoothly, while conversion of the corresponding β-arylalanines was poor or non-existent. In the range of 7-9 pH had no
significant influence on conversion of racemic α- or β-(thiophen-2-yl)alanines, whereas increasing the concentration of
ammonia (ammonium carbonate from 50 to 1000 mM) inhibited the isomerization progressively and decreasing amount
of the by-product, i.e. (E)-3-(thiophen-2-yl)acrylic acid, was detected. In all cases, the high ee values of the products
indicated excellent enantiomer selectivity and stereospecificity of the isomerization except for (S)-2-nitro-α-phenylalanine
(ee 92%) from the β-isomer. Substituent effects were rationalized by computational modelling revealing that one of the
main factor controlling biocatalytic activity was the energy difference between the covalent regioisomeric enzyme-
substrate complexes.
An attractive method for the synthesis of enantiopure, non-
natural β-amino acids is based on the use of phenylalanine
2,3-aminomutase (PAM). According to their stereochemical
preference there are two kinds of phenylalanine 2,3-amino-
Introduction
Nowadays there is an ever increasing demand for optically
pure β-amino acids mainly by the pharmaceutical industry and
peptide syntheses.1 In particular, the biological characteristics
of the β-amino acids, along with their use as precursors of
various heterocycles and as chiral auxiliaries in
enantioselective syntheses have aroused lively interest for
their chemistry. This induced rapid development of synthetic
procedures for the preparation of enantiopure β-amino acids
and their congeners.2 Until recently most of the biocatalytic
approaches for them relied on kinetic resolution with
hydrolytic enzymes, such as lipases2, acylases3 and
hydantoinases.4
mutases (PAMs), the one of plant origin (EC 5.4.3.10) is
producing (R)-β-phenylalanine5, while the other one is of
bacterial origin (EC 5.4.3.11) and is leading to (S)-β-phenyl-
alanine.6,7 Both are members of the class I lyase-like family
also including tyrosine 2,3-aminomutase (TAM, EC 5.4.3.6)8,
phenylalanine ammonia-lyase (PAL, EC 4.3.1.24 and EC
4.3.1.25)9, tyrosine ammonia-lyase (TAL, EC 4.3.1.23 and EC
4.3.1.25)10, and histidine ammonia-lyase (HAL, EC 4.3.1.3).11 All
of them utilize the same protein-derived prosthetic group,
3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) (Fig. 1A),
formed autocatalytically in the active site from an XSG motif
which is typically Ala-Ser-Gly.11 Less frequently MIO could be
formed from a Thr-Ser-Gly (in PaPAM and in SmPAM), a Ser-
Ser-Gly (in HAL from Fusobacterium nucleatum) or a Cys-Ser-
Gly (in HAL from Streptomyces griseus) motif as well.12
a.Biocatalysis and Biotransformation Research Group, Babeș-Bolyai University of
Cluj-Napoca, Arany János str. 11, RO-400028 Cluj-Napoca, Romania.
e-mail: paizs@chem.ubbcluj.ro
The broad range of aromatic and heteroaromatic amino acids
b.Department of Organic Chemistry and Technology, Budapest University of tolerated as substrates by these enzymes was also exploited
Technology and Economics, Műegyetem rkp. 3, H-1111 Budapest, Hungary. e-
for the preparation of a wide range of non-natural aryl and
heteroaryl α- and β-amino acids.13
mail: poppe@mail.bme.hu
c. Agricultural Institute, Centre of Agricultural Research, Hungarian Academy of
Sciences, Brunszvik u. 2, H-2462 Martonvásár, Hungary.
d.Institute of Organic Chemistry, Karlsruhe Institute of Technology, Richard-
Willstätter-Allee, D-76128 Karlsruhe, Germany.
PAM from Taxus canadensis (TcPAM) forming (R)-β-
phenylalanine was used for the partial biotransformation of
(S)-α-phenylalanine and its derivatives into their (R)-β-
isomer5,14, while the closely related Taxus chinensis (TchPAM)
e. SynBiocat Ltd, Lázár deék u 4/1, H-1173 Budapest, Hungary.
† This paper is dedicated to Professor Albert Eschenmoser on the occasion of his
90th birthday.
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as biocatalyst was employed in the enantioselective ammonia further various chiral phenylalanine derivatives19 as well as in
addition to (E)-cinnamate producing a mixture of enantiopure the synthesis of the anticancer drug Taxol.20
(S)-α- and (R)-β-phenylalanine.15 In a later study, significant
The crystal structure of PaPAM complexed with
shift of the regioisomeric preference towards the β-isomers phenylalanine to its active site, supported reaction
a
was achieved by site directed mutagenesis.16 Wanninayake et mechanism proceeding through two N-MIO containing
al. have exploited some unnatural amino acids as amino group intermediates.6 This was also in agreement with QM/MM
donors.17 The amino group of these substrates was transferred calculations on TAL21 and PAL22 supporting ammonia
by TcPAM intermolecularly to another arylacrylate skeleton to elimination via an N-MIO intermediate and suggesting the
form mixtures of α- and β-arylalanines.
formation of similar N-MIO complexes as a common feature of
Acting on α-phenylalanines the (S)-isomer-preferring PAM the mechanism for all MIO-enzymes.21 According to these
(EC 5.4.3.11) forms (S)-β-phenylalanine transposing exclusively propositions, the steps starting from either α- or
the amino group of the (S)-α-phenylalanine to give (S)-β- β-phenylalanine are quite similar such as: (i) formation of a
phenylalanine (Fig. 1B).6 Among the known members of the covalent enzyme-substrate complex via Michael addition of
PAM family producing (S)-β-phenylalanine are AdmH from the amino group of the substrate onto MIO, (ii) ammonia
Pantoea agglomerans (PaPAM)6 and EncP from Streptomyces elimination from the covalent N-MIO intermediate resulting in
maritimus (SmPAM).7 These enzymes have important
applications in the preparation of the antibiotic Andrimid18, mechanism proceeds further with (iii) ammonia re-addition
a cinnamate binding intermediate state (Fig. 1A). The
A)
(S)- phenylalanine
(S)- -phenylalanine
Tyr1-O
Tyr1-O
H
H
H
H
OOC
OOC
H
H
(E)-cinnamate
H3N
NH3
Tyr2-O
Tyr2-O
H
OOC
H
O
O
N
N
N
N
MIO
Tyr1: Tyr78
Tyr2: Tyr320
Tyr1-O
H
Tyr1-O
H
Tyr1-OH
H
H
H
H
OOC
H
OOC
OOC
H
NH2
H2N
N
NH2
Tyr2-OH
Tyr2-OH
Tyr2-OH
O
O
O
N
N
N
N
N
B)
C)
PaPAM
NH3
COO
NH3
COO
NH3
(EC 5.4.3.11)
COO
Ar
Ar
COO
Ar
Ar
(E)-arylacrylate
(R)- -arylalanine
(S)- -arylalanine
(S)- -arylalanine
PaPAM
NH3
NH3
COO
Ar
COO
Ar
(EC 5.4.3.11)
COO
COO
Ar
Ar
NH3
(R)- -arylalanine
(S)- -arylalanine
(E)-arylacrylate
(S)- -arylalanine
Figure 1 The proposed mechanism of action of PaPAM (A): starting from either direction with the cooperation of two catalytically essential
tyrosine residues (Tyr1 and Tyr2) and the MIO prosthetic group, N-MIO intermediates of α- or β-phenylalanine are formed which interconvert
by α
↔β migration of the amino group involving a postulated (E)-cinnamate intermediate. PaPAM-catalysed kinetic resolutions starting from
(B
) racemic α-arylalanines or (C
) racemic β-arylalanines result in the opposite enantiomers as products vs. unreacted enantiomers.
2 | RSC Advances, 2016, 00, 1-9
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and (iv) the release of the product. Occasionally, cinnamic acid
can appear as a by-product (Fig. 1A), supposedly due to
intermittent opening of the Tyr78-containig loop resulting in a
leak from the main cycle at the cinnamate binding
intermediate state.
Table 1 Composition of mixtures obtained from (±)-α-arylalanines
rac-1a-l with PaPAM (after 20 h incubation)
a
Substrate (Ar)
x1
x(S)-2
0.28
0.15
0.18
0.07
0.19
x3
rac-1a (phenyl)
0.66
0.74
0.82
0.8
0.77
0.77
1.00b
0.79
0.94
1.00b
0.78
0.94
0.04
SmPAM, described earlier as a lyase, has been shown lately
to be closely related to PaPAM (63% overall sequence identity
and 76% sequence similarity).7 More recently, Weise et al.
investigated SmPAM in the context of ammonia addition to
several aryl-substituted (E)-cinnamic acid analogues.23 They
found that SmPAM converted a range of arylacrylates to a
mixture of (S)-α- and (S)-β-arylalanines. The enzyme exhibited
variable regioselectivity, much affected by ring substituents,
but introduction of certain active site mutations could shift
regioselectivity in either direction. However, in SmPAM-
catalysed isomerization of (S)-α- and (S)-β-arylalanines the
enantioselectivity was incomplete in many cases.23
rac-1b (thiophen-2-yl)
rac-1c (4-bromophenyl)
rac-1d (2-fluorophenyl)
rac-1e (3-fuorophenyl)
rac-1f (4-fluorophenyl)
rac-1g (2-chlorophenyl)
rac-1h (3-chlorophenyl)
rac-1i (4-chlorophenyl)
rac-1j (2-nitrophenyl)
rac-1k (3-nitrophenyl)
rac-1l (4-nitrophenyl)
0.11
c
0.13
0.04
0.20
0.03
b
b
0.2
0.02
c
0.06
b
b
c
c
0.22
0.06
aee >98% when not stated otherwise; b no reaction was observed;
c not observed
Substrate specificity of PaPAM was tested with a wide range
of aromatic and heteroaromatic (S)-α-arylalanines.19 Electronic
and steric effects of substituents at the aromatic ring
significantly influenced both catalytic efficiency and the
formation of arylacrylates as by-products. It was observed that
3-substituted (S)-α-phenylalanines were transformed faster
than the 2- or 4-substituted isomers. In order to explain these
x1, x(S)-2 and x3 represent the relative molar fractions of the
reaction components as determined by 1H-, 19F-NMR
measurements
reactions, using rac-β-phenylalanines (Fig. 1C) or rac-α-
phenylalanines (Fig. 1B) as substrates. Therefore the results
after 20 h show the final product distribution near to the
equilibrium state, after longer reaction times further product
formation was not observed.
observations, computational analysis of substrate−PaPAM
structural interactions was performed by substrate docking
studies.19 Recently it was shown that recombinant whole cell
E. coli expressing PaPAM could also produce enantiopure (S)-
β-arylalanines from (S)-α-arylalanines.24 Worth noting, that
PaPAM failed to catalyse the transformation of several 2-
substituted (S)-α-phenylalanines studied.19,24
Transformation of (±)-α-arylalanines(rac-1a-l) with PaPAM
According to previous results19, PaPAM failed to catalyse
transformation of 2-substituted (S)-α-phenylalanines bearing
large electron withdrawing substituents. Our study starting
from (±)-α-arylalanines rac-1a-l (Fig. 2A) confirmed the validity
of this observation also for the racemic mixtures (Table 1).
In the presence of PaPAM, no product could be detected
Results and discussion
The time course of the reaction (Supplementary material)
showed that after a relatively short timp period (2-6 hours) an
equlibrium state is reached in case of both enzymatic
with (±)-α-(2-chlorophenyl)alanine (rac-1g
)
or (±)-α-(2-
(±)-α-(2-
nitrophenyl)alanine (rac-1j), while with
Figure 2 PaPAM-catalysed transformation of (A) (±)-α-arylalanines rac-1a-l and (B) (±)-β-arylalanines rac-2a-l
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fluorophenyl)alanine (rac-1d
) being substituted with the Importantly, high enantiopurity of the products [>98% ee for
smallest halogen atom moderate mutase-activity was (S)-2a-f,h,i,k,l] indicated high enantioselectivity of the PaPAM-
observed resulting in the formation of enantiopure (S)-β-(2- catalysed isomerization in the α β-direction. The observed
fluorophenyl)alanine [(S)-2d]. high stereoselectivity of the PaPAM-catalysed isomerization of
→
Conversion of the (±)-3-substituted-α-phenylalanines the (S)-α- and (S)-β-arylalanines was a major advantage
(rac-1e,h,k) with PaPAM within 20 h was moderately faster compared to the incomplete stereoselectivity of SmPAM-
than that of the 2-substituted substrate rac-1d, but still slower catalysed isomerizations.23
than with (±)-α-phenylalanine (rac-1a).
Comparison of the conversions of the (±)-4-substituted-α-
Transformation of (±)-β-arylalanines (rac-2a-l) with PaPAM
phenylalanines (rac-1c,f,i,l) with PaPAM for 20 h indicated that
In order to explore the potential of kinetic resolutions of
with (±)-4-fluoro- (rac-1f) and (±)-4-bromo-α-phenylalanine
(±)-α- and β-arylalanines for the preparation of antipodal
(rac-1c) about 1.2 times higher conversions were attained than
products, we extended our study to the reactions of
with (±)-4-chloro-(rac-1i
(rac-1l).
)
or (±)-4-nitro-α-phenylalanine
(±)-β-arylalanines rac-2a-l (Fig. 2B and Table 2). The similar
time course profiles of the product formation in PaPAM
Reactions of (±)-α-arylalanine bearing thiophen-2-yl group as
a small heteroaromatic moiety (rac-1b) with PaPAM were also
investigated. It was found that the conversion of
catalyzed
reactions
from
(S)-β-phenylalanine
and
rac-β-phenylalanine (Supplementary material) supported that
the unreactive (R)-β-phenylalanine did not act as a significant
inhibitor. In contrast to the (±)-2-substituted α-phenylalanines
with large substituents (rac-1g,j), which were apparently not
accepted as substrates by PaPAM, all the (±)-2-substituted β-
phenylalanines in the present study (rac-1d,g,j) were smoothly
transformed. On the other hand, sluggish or no conversion was
observed with PaPAM using as substrates (±)-3- and (±)-4-
substituted β-phenylalanines bearing bulky electron
withdrawing substituents (rac-2c,h,i,k,l).
(±)-α-(thiophen-2-yl)alanine (rac-1b
(±)-α-phenylalanine (rac-1a).
)
was similar to
In an earlier study with (S)-1a, negligible ammonia-lyase
activity was predicted for PaPAM at low temperatures (under
30 °C).7 In another study19 and also in our present one,
however, formation of significant amount of (E)-arylacrylates
was observed in many cases
(3a,b,d,e,f,h,l in Table 1)
indicating substantial lyase-activity of PaPAM. Note that with
(±)-4-bromo- (rac-1c), (±)-4-chloro (rac-1i) and (±)-3-nitro-α-
phenylalanine (rac-1k) as substrates no such activity was
observed. Relatively high amounts of arylacrylates were
formed in reactions with substrates bearing the smallest
aromatic moieties i.e. with (±)-α-phenylalanine (rac-1a),
The conversions of (±)-β-(thiophen-2-yl)alanine (rac-2b) and
(±)-3-fluoro-β-phenylalanine (rac-2e) with PaPAM were higher
than that of (±)-β-phenylalanine (rac-2a). The (±)-4-fluoro-β-
phenylalanine (rac-2f
) was converted similarly as (±)-β-
phenylalanine (rac-2a), while (±)-4-bromo-β-phenylalanine
(rac-2c) was transformed to the α-isomer (S)-1c and a small
(±)-α-(thiophen-2-yl)alanine (rac-1b
)
and (±)-2-fluoro-α-
phenylalanine (rac-1d).
amount of 4-bromocinnamate (3c
) at significantly lower
Results with the (±)-α-arylalanines (rac-1a-l) demonstrated
that mutase and/or lyase activity of PaPAM was much affected
by the nature of the aromatic moiety of the substrates.
conversion than rac-2a. In all cases, except for (±)-2-nitro-β-
phenylalanine (rac-2j), reactions catalysed by PaPAM
proceeded with the formation of the enantiopure (S)-α-isomer
[(S)-3a-g] and some arylacrylate (3a-g). Transformation of
(±)-2-nitro-β-phenylalanine (rac-2j) was an exception owing to
Table
2 Composition of the mixtures obtained from (±)-β-
arylalanines rac-2a-l with PaPAM (after 20 h incubation)
the absence of the by-product
(3j) and incomplete
a
stereoselectivity (ee(S)-1j= 92%).
Substrate(Ar)
x2
x(S)-1
0.29
0.22
0.08
0.35
0.25
0.23
x3
rac-2a (phenyl)
0.70
0.73
0.91
0.63
0.67
0.76
0.54
1.00b
1.00b
0.85
1.00b
1.00b
0.01
0.04
0.01
0.02
0.08
0.01
rac-2b (thiophen-2-yl)
rac-2c (4-bromophenyl)
rac-2d (2-fluorophenyl)
rac-2e (3-fuorophenyl)
rac-2f (4-fluorophenyl)
rac-2g (2-chlorophenyl)
rac-2h (3-chlorophenyl)
rac-2i (4-chlorophenyl)
rac-2j (2-nitrophenyl)
rac-2k (3-nitrophenyl)
rac-2l (4-nitrophenyl)
Effects of pH and ammonia concentration on the PaPAM-catalysed
isomerization of (±)-α- and β-(thiophen-2-yl)alanine (rac-1b and
rac-2b)c
Prompted by the fact that conversions from (±)-α- and (±)-β-
(thiophen-2-yl)alanine (rac-1b and rac-2b) with PaPAM were
similar to those from the natural substrates i.e. (±)-α- and β-
phenylalanine (rac-1a and rac-2a) but more of the by-product
[(E)-3-(thiophen-2-yl)acrylate, 3b] was formed (Table 1 and
Table 2), transformations of these substrates were studied in
more detail by varying pH or ammonia concentration. An
alteration of pH of the buffer solution in the range of 7–9
[at 100 mM (NH4)2CO3] was indifferent to conversion (data not
shown), unlike changing the (NH4)2CO3 concentration in the
range of 50–1000 mM (at pH 8) which significantly influenced
product compositions (Table 3).
0.40
b
0.06
b
b
b
d
b
b
0.15c
b
b
aee >98% when not stated otherwise; b no reaction was observed;
cee= 92% d not observed
x2, x(S)-1 and x3 represent the relative molar fractions of the
reaction components as determined by 1H-, 19F-NMR
measurements
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isomerization and ammonia elimination as side reaction in
both directions of the reaction.
Table 3 Composition of the reaction mixtures obtained from (±)-
α- or (±)-β-(thiophen-2-yl)alanine (rac-1b or rac-2b) with PaPAM
at various ammonium carbonate concentrations (after 20 h)
Computational modelling of PaPAM-catalysed isomerizations
c(NH4)2CO3
(mM)
50
100
200
1000
50
100
200
1000
To rationalize the effect of substituents of aromatic and
Substrate
x1b
x2b
x3b
heteroaromatic (±)-α- and β-arylalanines (rac-1a-l and rac-2a-l
)
rac-1b
rac-1b
rac-1b
rac-1b
rac-2b
rac-2b
rac-2b
rac-2b
0.68
0.68
0.72
0.76
0.19
0.27
0.23
0.13
0.17
0.17
0.16
0.12
0.76
0.69
0.75
0.84
0.15
0.15
0.12
0.12
0.05
0.04
0.02
0.03
on PaPAM-catalysed isomerizations computational and
statistical analysis was performed based on modelling and
comparison of the N-MIO states (S)-1a-i,k,lN-MIO and
(S)-2a-i,k,lN-MIO formed from the corresponding (S)-α- and (S)-
β-arylalanines. Because of the experimentally observed
stereospecificity of isomerizations, in the computations only
the (S)-enantiomers were considered. Due to the mixed
mechanism indicated by the incomplete enantioselectivity in
the PaPAM-catalysed reaction of rac-2j, data calculated for the
reactions of (S)-α- and (S)-β-2-nitrophenylalanine [(S)-1j and
(S)-2j] were omitted from the comparison of reactivities.
x1b, x2b and x3b represent the relative molar fractions of the
reaction components as determined by 1H-NMR measurements
Analysis of the PaPAM-catalysed reactions of (±)-α-
In molecular mechanics the bonded terms measure the
strain compared to a hypothetical zero-point energy. Thus
potential energies derived from molecular mechanics
calculations cannot be compared directly if the molecular
structures to be related do not have exactly the same atom
connectivity. In this modelling study it was assumed that for all
investigated compounds the common alanine part of α- and β-
arylalanines underwent in the corresponding N-MIO
intermediates similar structural changes6,21 (Fig.1). Therefore,
it was possible to compare the difference of the energies
calculated for the N-MIO intermediates from the (S)-α- and (S)-
β-arylalanines [(S)-1b-i,k,lN-MIO and (S)-2b-i,k,lN-MIO] relative to
the corresponding values obtained for (S)-α- and (S)-β-
(thiophen-2-yl)alanine (rac-1b
)
at various (NH4)2CO3
concentrations with 20 h incubation showed that increasing
(NH4)2CO3 concentration above 100 mM resulted in decreasing
conversion to both (S)-β-(thiophen-2-yl)alanine (S)-2b and the
elimination product 3b. At 1000 mM(NH4)2CO3 concentration
both (S)-β-(thiophen-2-yl)alanine (S)-2b and arylacrylate3b
formation dropped to 12%. Interestingly, when starting from
(±)-β-(thiophen-2-yl)alanine (rac-2b), the best conversion to
(S)-α-(thiophen-2-yl)alanine (S)-1b (27%) was achieved at
100 mM buffer concentration. At higher (NH4)2CO3
concentrations smaller conversions were observed.
Study of the dependence of the PaPAM-catalysed reactions
of (±)-α- and β-(thiophen-2-yl)alanine (rac-1b and rac-2b
,
phenylalanine [(S)-1aN-MIO and (S)-2aN-MIO], explicitly: (E(S)-2-E(S)- )-
1
respectively) on (NH4)2CO3 concentration indicated that
elevated ammonia concentrations lowered both the rate of
(E(S)-2a-E(S)-
)
,
thus cancelling the hypothetical terms
1a
representing the difference in heat of formation for the α- and
Table 4 Differences of conversions of the PaPAM-catalysed isomerizations from (±)-α- and β-arylalanines (rac-1a-l and rac-2a-l), substrate
categories and relative energy differences of the regioisomeric N-MIO intermediates [(S)-1a-lN-MIO and (S)-2a-lN-MIO
]
Substrates
Difference of conversionsa
Categoryb
Energy difference
(kcal/mol)c
0.0
(S)-1,2a
(S)-1,2b
(S)-1,2c
(S)-1,2d
(S)-1,2e
(S)-1,2f
(S)-1,2g
(S)-1,2h
(S)-1,2i
(S)-1,2j
(S)-1,2k
(S)-1,2l
-0.05
-0.01
-0.12
0.19
0.09
0.01
0.46
-0.16
E
E
A
B
E
E
B
A
6.1
-3.6
1.8 (1.0)
-4.1
0.2
3.1 (0.6)
-3.0
-2.8
-0.10
A
d
d
d
-7.2 (-2.0)
-6.7
-0.26
-0.11
A
A
a Difference of conversions is defined as: (1-x2)-(1-x1), where x1, x2 are the corresponding molar fraction values listed in Table 1 and Table 2. b Categories
based on regioisomeric preference are defined as follows: A: (1-x2)-(1-x1) < -0.09; E: -0.09 ≤(1-x2)-(1-x1) ≤ 0.09; B: (1-x2)-(1-x1) > 0.09. cEnergy difference
of N-MIO states (S)-1b-lN-MIO and (S)-2b-lN-MIOis defined and normalized for (S)-1aN-MIO and (S)-2aN-MIO as: (E(S)-2-E(S)-1)-(E(S)-2a-E(S)-1a). For compounds with
different ring orientations in their lowest energy conformations of the α- and β-N-MIO intermediate states, energy values are selected for structures
consistent with the reaction of lower or no conversion. Energy differences pertinent to the other direction are presented in parentheses. d Due to the
mixed mechanism indicated by the incomplete stereoselectivity in the reaction of rac-2j with PaPAM, data for (S)-1jN-MIO and (S)-2jN-MIO were not
included.
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A)
B)
Figure 3 Comparison of the arrangements of regioisomeric N-MIO intermediates in their PaPAM-catalysed transformations.
) N-MIO intermediates of (S)-α-(3-chlorophenyl)alanine (solid model) and of (S)-β-(3-chlorophenyl)alanine (transparent model) [(S)-1hN-MIO
and (S)-2hN-MIO]; ( ) the N-MIO intermediates of (S)-α-(2-chlorophenyl)alanine (solid model) and (S)-β-(2-chlorophenyl)alanine (transparent
model) [(S)-1gN-MIO and (S)-2gN-MIO]. Panel depicts a case where the lowest energy conformations of the two regioisomers adopt similar
orientations of the aromatic ring. Panel illustrates a case where in the lowest energy conformations of the two regioisomers the aromatic
rings occupy different orientations (differing by a flip of 180°). Images were created using PyMOL.25
(A
B
A
B
β-alanine part (Table 4).
further nonparametric tests – having lower statistical power
Our molecular modelling studies based on an exhaustive than parametric methods but without the requirement of
search for possible conformations of the covalently bound normal distribution of data – were also performed. The
N-MIO intermediates of the (S)-α- and (S)-β-arylalanines Kruskal-Wallis ANOVA and Mann-Whitney U tests resulted only
[(S)-1b-i,k,lN
and (S)-2b-i,k,lN-MIO], indicated that unlike the somewhat higher p values (0.100 and 0.053, respectively) than
-MIO
reactions of the majority of the regioisomers the threshold. Moreover, the median test showed also
[(S)-1,2a-c,e,f,h-j,l and significant differences between the categories. Overall, the
see Fig. 3A25 for (S)-1hN
;
-MIO
(S)-2hN-MIO], in certain pairs of isomers [(S)-1,2d,(S)-1,2g statistical tests supported the finding that categories A and B
and(S)-1,2k] the arrangements of the aromatic ring in the are truly separate.
lowest energy conformations of the enzyme-bound substrate
complexes were dissimilar and differing by a 180° flip [see Fig.
3B for (S)-1gN
and (S)-2gN-MIO]. Assuming that in the N-MIO
-MIO
complexes in the tightly packed active site of PaPAM there is
no room for a flip neither of the complete arylacrylate
molecule nor of the aromatic ring only, it can be predicted that
in such cases one of the two N-MIO intermediate states is not
in its lowest energy state. Thus the calculated lowest energy
differences in such cases should be higher, corresponding to
the allowed arrangements without involving
hindered ring flip (Table 4).
a sterically
Statistical analysis of the experimental and computational
data revealed that compounds (S)-1,2a-i,k,l could be classified
into three categories (A, E and B, in Table 4) based on the
difference of conversions [defined as: (1-x2)-(1-x1), where x1, x2
are the corresponding molar fraction values listed in Table 1
and Table 2]. The three categories: A: (1-x2)-(1-x1) < -0.09;
E: -0.09 ≤ (1-x2)-(1-x1) ≤ 0.09 and B: (1-x2)-(1-x1) > 0.09 could be
correlated with the regioisomeric preferences (Table 4, Figure
4). One-way ANOVA test – treating the categories (Table 4) as
the independent and the conversion differences (Table 1 and
2) as the dependent variables – indicated significant difference
between categories A and B at the α=0.050 level. In addition,
Figure 4 Box-whisker plot depicting the energy differences of the
corresponding N-MIO intermediates [(S)-1a-i.k.lN-MIO and (S)-2a-
I,k,lN-MIO] as a function of the catalytic activity categories (A, B and
E). Legend – square: median; boxes: span between the lower and
upper quartile, whiskers: span of the entire set.
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The computational study extended with statistical analysis isomerizations were stereospecific giving mixture of unreacted
revealed that the cases when one of the regioisomers was (R)-α- or (R)-β-arylalanines with an enantiomeric excess
converted much faster (or is the only one to be converted) the depending on conversion, and enantiopure (S)-α- or β-
energetics of the regioisomeric N-MIO-type enzyme-substrate arylalanines as products, along with various amounts of
complexes was one of the most important factors governing arylacrylate as by-product. Computational and statistical
the outcome of the reaction. In the case of the intermediate analysis revealed significant correlation between the
group (category E) involving small aromatic moieties energetics of the N-MIO intermediate states forming from the
contribution of the steric effects are much less pronounced.
(S)-α- or β-arylalanines and the regioisomeric preferences of
The computational results listed in Table 4 suggest that PaPAM in case of substrates with bulky electron withdrawing
the reactions of 2-substituted α-phenylalanines [(S)-1d,g and substituents on the aromatic ring. In several cases “hysteresis”
(S)-2d,g: category B in Table 4 and Figure 4] are strongly was postulated: the conformation (thus energy) of a given
disfavoured because the energy calculated for the α-N-MIO regioisomeric N-MIO intermediate formed from the given
intermediates [(S)-1d,gN
] is much lower than that regioisomeric substrate differed from that conformation which
-MIO
calculated for the corresponding β-N-MIO intermediates [(S)- resulted in the given regioisomer as product.
2d,gN-MIO]. This was in full agreement with experimental
observations indicating slow or no reaction from the
Experimental
(±)-2-substituted α-phenylalanines (rac-1d,g,j) with PaPAM, in
contrast to high conversions from 2-substituted
(±)-β-phenylalanines (rac-2d,g,j) under the same conditions.
The situation for the 3- and 4-substituted phenylalanines with
bulky substituents [(S)-1c,h,i,k,l and (S)-2c,h,i,k,l: category A in
Table 4 and Figure 4] was the opposite. In good correlation
with the experimental regioisomeric preferences, much lower
energies were calculated for the β-N-MIO intermediates [(S)-
Reagents and analytical methods
The starting materials were purchased from Sigma-Aldrich
(St. Louis, MO, USA) or Carl Roth (Karlsruhe, Germany) and
used without purification. Solvents were purified and dried by
standard methods. The racemic amino acids rac-1a-l rac-2a-l
and the α,β-unsaturated acids 3a-l were synthesized using
published methods.26
2c,h,i,k,lN
]
than for the corresponding α-N-MIO
-MIO
intermediates [(S)-1c,h,i,k,lN-MIO]. This could explain sluggish or
no conversion of β-arylalanines containing bulky substituents
at positions 3 or 4 (rac-2c,h,i,k,l) with PaPAM and much higher
conversions of the α compounds (rac-1c,h,i,k,l) under the
same conditions. In addition, the energy differences
corresponding to the direction of reaction with higher
conversions and assuming a disallowed flip of the aromatic
ring (Table 4, values in parentheses) showed remarkable
differences compared to the sterically non-restricted values
and the hindered flip of the aromatic ring further amplified the
difference in reactivity mentioned before. Because of the
hindered flip, in case of certain regioisomeric amino acid pairs
the aromatic ring in a given N-MIO intermediate adopts
different conformation depending on whether the actual
N-MIO intermediate is at the substrate side [step ii) in our
postulated mechanism, Fig. 1A] or at the product side [step iii)
in our postulated mechanism, Fig. 1A].
The 1H- and 19F-NMR spectra were recorded at 21 °C on
Bruker spectrometers operating at 400 MHz, 101 MHz and 600
MHz, 151 MHz, respectively. Enantiomer separations were
obtained on Agilent 1260 HPLC instrument using either
Chiralpak® ZWIX(+) (4 mm × 250 mm) column and a mixture of
methanol (containing 100 mmol/L formic acid and 50 mmol/L
diethylamine), acetonitrile and water in proportion of 49:49:2
(v/v/v) as eluent at a flow rate of 1 mL/min or Crownpak®
CR-I(+) column (150 × 3.0 mm × 5 µm) and a mixture of HClO4
solution (3.6 g/L, pH 1.5):acetonitrile as mobile phase at a flow
rate of 0.4 mL/min. NMR spectra and HPLC data are presented
as Supplementary material.
Expression and purification of PaPAM
The gene of PAM from Pantoea agglomerans (encoding 541
AAs – Uniprot code: Q84FL5) was optimized to the codon
usage of E. coli. The 1626 bps long synthetic gene was
produced and cloned into pET-19b vector using the XhoI and
Bpu1102I cloning sites. The recombinant PaPAM carrying an
N-terminal (His)10-tag was produced in E. coli BL21(DE3)pLysS
cells. For the expression step a colony of the transformed
plasmid was grown overnight at 37 °C in 5 mL of LB medium
containing carbenicillin (50 µg/mL) and chloramphenicol (30
µg/mL). The overnight culture was added to LB medium (0.5 L)
in an Erlenmeyer flask and grown at 37 °C until OD600 reached
0.7 - 0.8. Then the temperature was lowered to 25 °C and the
cells were induced by the addition of IPTG (1 mM). The culture
was shaken at 220 rpm at 25 °C for 19 h longer. All of the
subsequent procedures were carried out in an ice-bath. The
cells were harvested by centrifugation (25 min, 5000 × g) and
Conclusion
According to our results, regioselectivity and activity of
PaPAM towards α- and β-arylalanines are mainly influenced by
the nature of the aromatic moiety of the substrates. PaPAM
catalyses the synthesis of the corresponding (S)-β-enantiomer
from racemic 3- and 4-substituted α-phenylalanines more
smoothly than that starting from racemic 2-substituted α-
phenylalanines, the latter being poor or no substrates of the
enzyme. Contrarily, racemic 2-substituted β-phenylalanines
were good substrates and provided the (S)-α-enantiomers
smoothly while the racemic 3- or 4-substituted β-
phenylalanines were almost no substrates. Importantly, in all
but one case (for racemic 2-nitro-β-phenylalanine), the
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re-suspended in 50 mL of lysis buffer (150 mM NaCl, 50 mM
The homotetrameric X-ray structure of PaPAM [PDB ID:
TRIS, pH 7.5) supplemented with DNAse, RNAse, Lysosyme, 3UNV]6 was completed and adjusted using the Protein
PMSF (2 mM) and an EDTA-free protease-inhibitor cocktail. Preparation Wizard29 in four steps: (i) hydrogen atoms were
The cells were then lysed by sonication and the cell debris was added and bond orders were assigned, (ii) artefacts of the
removed by centrifugation (10000 × g, 30 min).
protein crystallization procedure were removed, except for
The proteins were purified on a column filled with nickel- two phosphate ions, (iii) hydrogen bond network, tautomeric
nitrilotriacetic acid agarose gel (Ni-NTA) following the states, side chain conformations of selected amino acids and
manufacturer’s protocol.27 The expressed protein was eluted ionization states were determined and optimized
from the column with imidazole (500 mM in Low Salt Buffer, corresponding to the experimental assay conditions and (iv) a
pH 7.5). The purity of the protein in the eluted fractions was constrained minimization was performed. Protein pKa were
verified by SDS-PAGE analysis. The protein fractions were predicted using PROPKA.30 In the further modelling process for
dialyzed against phosphate-buffered saline (50 mM) at 4 °C the N-MIO intermediates, in accordance with to the proposed
followed by concentration by centrifugal ultrafiltration (using mechanism (Fig. 1), Tyr78 was set deprotonated and Tyr320
vertically-oriented ultrafiltration membrane VIVASPIN 10,000 protonated. Further details on the computational methods and
MWCO, 5000 × g, 4 °C, to final concentration of 3-5 mg/mL). the model will be published in a forthcoming paper.
The concentration of PaPAM in the final solutions was
determined by Bradford’s method.28
The refined and completed X-ray structure served as a
starting point to create an overall protein model
corresponding to the experimental assay conditions. The
buffer solution solvated model was created by the Desmond
program suite.31 The PaPAM model was solvated explicitly
with water and additional ions were added with respect to the
experimental assay conditions. The buffer solvated model was
then equilibrated with a slightly modified default equilibration
protocol, applying harmonic constraints to the Cartesian
coordinates of protein heavy atoms. A spherical model of the
active site with a radius of 27 Å and centred on the exocyclic
methylene carbon of the MIO prosthetic group of chain C was
cut off and capped with acetyl and N-methylamino groups.
N-MIO type covalent complexes of our substrates (S)-1a-l
and (S)-2a-lwere created by our induced-fit covalent docking
protocol. This involved the creation of initial conformations of
compounds (S)-1a-l and (S)-2a-l by docking with Glide program
suite32 into a modified and artificially enlarged active site in
which the residues Leu216, Ile219, Leu104, Val108, Met84,
Leu421, Leu171, and Phe428 were exchanged to Ala residues,
further the MIO prosthetic group was reduced to Thr + Gly and
three water molecules in the active site were removed.
After having docked into the enlarged active site, all side
chains and the MIO group were restored, a covalent bond
between the nitrogen atom of the amino group and the
exocyclic carbon of MIO was created, the covalently bound
ligands and the residues in close proximity to them were
minimized and finally, redundant conformations were
eliminated with MacroModel.33 During restoring the mutated
side-chains, conformations of several active site residues were
predicted with Prime.34
PaPAM-catalysed biotransformations of (±)-α- and β-arylalanines
Into the solution of the substrate (rac-1a-f,h,i,k,l and rac-2a-
g,j, 4 mg) in (NH4)2CO3 buffer (100 mM, pH 8.0, 2 mL), PaPAM
(1.6 mg) was added and the reaction mixture was stirred at
room temperature for 20 h. For HPLC measurements reaction
samples (30 µL) were taken and the enzyme was precipitated
with 30 µL MeOH. After filtration, the samples were diluted
with the corresponding mobile phase and injected on HPLC.
Prior to 1H- and 19F-NMR investigations the reaction was
quenched with methanol, followed by filtration and
evaporation of the solvent in vacuum. Deuterated sodium
hydroxide (2 % NaOD) solution was added and the spectrum
(Supporting Information) was recorded at room temperature.
Biotransformation of (±)-α- or β-(thiophen-2-yl)alanine (rac-1b or
rac-2b) under various conditions
pH change in the range of 7-9. Into the solution of the substrate
(rac-1b or rac-2b, 4 mg) in (NH4)2CO3buffer (pH 7.0, 8.0 or 9.0;
100 mM, 2 mL), PaPAM (1.6 mg) was added and the reaction
mixture was stirred at room temperature for 20 h. The reaction was
stopped by heating at 90 °C for 10 min. The solvent was
evaporated, the sample re-dissolved in NaOD solution (2%, 0.5 mL)
and analysed by 1H-NMR (data not shown).
Change of ammonium carbonate concentration in the range of 50-
1000 mM. Into the solution of the substrate (rac-1b or rac-2b, 4
mg) in (NH4)2CO3 buffer (50, 100, 200, or 1000 mM; pH 8.0; 2 mL),
PaPAM (1.6 mg) was added and the reaction mixture was stirred at
room temperature for 20 h. The reaction was stopped by heating at
90 °C for 10 min. Then the solvent was evaporated, the sample re-
dissolved in NaOD solution (2%, 0.5 mL) and analysed by 1H-NMR
(see Table 3).
After replacing the three active site water molecules
removed earlier, a final minimization in the 6Å proximity of the
covalently bound ligand using Prime34 resulted in the final
models and energies. OPLS2005 force field was applied in all
molecular mechanics calculations and simulations.
Statistical tests (one-way ANOVA, Kruskal-Wallis ANOVA,
median test and Mann-Whitney U tests) were carried out and
Figure 4 was created using Statistica.35 Non-significant Levene
and Shapiro-Wilk tests justified the use of one-way ANOVA.
The probability value of type I error (α) was chosen to be 0.05
in all the cases.
Molecular modelling of the covalent enzyme−substrate N-MIO
complexes in PaPAM
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Acknowledgements
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AV and GB contributed equally to this work. AV and NB
thank the financial support from the Sectoral Operational
Program for Human Resources Development 2007-2013, co-
financed by the European Social Fund (projects
POSDRU/159/1.5/S/137750 and POSDRU/159/1.5/S/132400).
The financial support provided by the Collegium Talentum
Research Program to NB and LP is also acknowledged. CP
thanks for financial support from the Romanian National
Authority for Scientific Research, CNCS – UEFISCDI (PN-II-
IDPCE-2011-3-0799). LP thanks for financial support from
Hungarian OTKA Foundation (NN-103242), from the Hungarian
Research and Technology Innovation Fund (KMR 12-1-2012-
0140) and from the New Hungary Development Plan (TÁMOP-
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