Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway

Article abstract of DOI:10.1002/anie.201311061

Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E₁cB elimination mechanism. All manner of things: A competing MIO-independent (MIO=4-methylideneimidazole-5-one) reaction pathway has been identified for phenylalanine ammonia lyases (PALs), which proceeds in a non-stereoselective manner, resulting in the generation of D-phenylalanine derivatives. The mechanism of D-amino acid formation is explored through isotopic-labeling studies and mutagenesis of key active-site residues.

Full text of DOI:10.1002/anie.201311061

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DOI: 10.1002/anie.201311061
Reaction Mechanisms
Phenylalanine Ammonia Lyase Catalyzed Synthesis of Amino Acids by
an MIO-Cofactor Independent Pathway**
Sarah L. Lovelock, Richard C. Lloyd, and Nicholas J. Turner*
Abstract: Phenylalanine ammonia lyases (PALs) belong to
a family of 4-methylideneimidazole-5-one (MIO) cofactor
dependent enzymes which are responsible for the conversion of
l-phenylalanine into trans-cinnamic acid in eukaryotic and
prokaryotic organisms. Under conditions of high ammonia
concentration, this deamination reaction is reversible and
hence there is considerable interest in the development of PALs
as biocatalysts for the enantioselective synthesis of non-natural
amino acids. Herein the discovery of a previously unobserved
competing MIO-independent reaction pathway, which pro-
ceeds in a non-stereoselective manner and results in the
generation of both l- and d-phenylalanine derivatives, is
described. The mechanism of the MIO-independent pathway is
explored through isotopic-labeling studies and mutagenesis of
key active-site residues. The results obtained are consistent with
amino acid deamination occurring by a stepwise E1cB
elimination mechanism.
atives.[5] These reactions are compatible with aqueous and
metal-free conditions and have the distinct advantage that no
protecting group chemistry is required. Additionally PALs
utilize readily available achiral cinnamic acid derivatives as
starting materials and they do not rely on external cofactors
or recycling systems, making them particularly suitable as
industrial biocatalysts.[6] This feature is highlighted by the
recent application of PAL in the synthesis of 2’-chloro-l-
phenylalanine on a ton scale, by DSM Pharma Chemicals.[7]
To allow structure-guided rational engineering for the
development of biocatalysts with desirable properties,
a detailed understanding of the enzymeꢀs catalytic mechanism
is highly advantageous. However the PAL catalytic mecha-
nism is still under investigation. PALs belong to a family of 4-
methylideneimidazole-5-one (MIO) cofactor dependent
enzymes, which also include histidine and tyrosine ammonia
lyases (HALs and TALs) and aminomutases (AMs), the latter
catalyzes the isomerization of a- and b-amino acids.[8] The
MIO cofactor is formed by an autocatalytic condensation
reaction of a conserved Ala-Ser-Gly motif. Mechanistic
studies suggest the presence of a covalent intermediate
formed by the addition of the substrate amine onto the
exocyclic methylene group of the cofactor (Figure 1).[9]
Optically active amino acids are fundamental building
blocks for the synthesis of a wide range of bioactive natural
products, pharmaceuticals, and agrochemicals. For example,
there is significant interest in the development of therapeutic
peptides and proteins which can contain both natural and
non-natural amino acids.[1] Non-proteinogenic amino acids
have also been used independently as drug molecules,
including Levodopa (l-dopa), the main drug used for the
treatment of Parkinsonꢀs disease.[2] Furthermore, asymmetric
synthesis frequently relies on amino acids as chiral starting
materials and auxiliaries.[3] The increasing demand to access
amino acids in optically pure form has led to the development
of a number of biotechnological approaches.[4] A particularly
attractive method for the synthesis of aromatic amino acids
involves the use of phenylalanine ammonia lyases (PALs),
a class of enzymes which catalyze the reversible regio- and
stereoselective hydroamination of trans-cinnamic acid deriv-
[*] S. L. Lovelock, Prof. N. J. Turner
School of Chemistry, University of Manchester
Manchester Institute of Biotechnology
131 Princess Street, Manchester, M1 7DN (UK)
E-mail: Nicholas.turner@manchester.ac.uk
Figure 1. Proposed E1cB mechanism for the PAL-catalyzed l-phenyl-
alanine deamination reaction.
Dr. R. C. Lloyd
Dr. Reddy’s Laboratories, Chirotech Technology Centre
410 Cambridge Science Park, Milton Road
Cambridge, CB4 0PE (UK)
Subsequent elimination of the amine to form cinnamic acid
is generally reported to occur by an E1cB mechanism,
although evidence in favor of an E2 elimination pathway has
also been presented.[10] An alternative Friedel–Crafts mech-
anism has been proposed on the basis that the elimination
mechanisms involve unfavorable abstraction of a non-acidic
proton from the b-position of the amino acid.[11] Nonetheless,
there is considerable evidence to support the formation of an
[**] Financial support from the Centre of Excellence in Biocatalysis,
Biotransformation and Biocatalytic Manufacture (CoEBio3:
www.coebio3.org) and a Royal Society Wolfson Research Merit
Award are gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201311061.
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amine–MIO intermediate, and the evidence includes crystal-
lographic data,[12] molecular mechanics calculations,[10,13] and
kinetic isotope studies.[14]
2h–m diminished significantly after prolonged reaction times
with all three PAL biocatalysts. These products all possess
electron-withdrawing substituents on the aromatic ring,
suggesting that PALs are able to catalyze the formation of
the d enantiomers of electron-deficient structures. d-amino
acid formation is especially prominent with substrates which
are able to stabilize a negative charge at the benzylic position,
for example, in the cases of 1k and 1m, which possess ortho
and para nitro substituents, respectively. The AvPAL-medi-
ated amination reaction of 1b, 1e–f, 1h–j, and 1l demon-
strated significantly higher enantioselectivity compared with
that of PcPAL and RgPAL under the reaction conditions
employed. Although the application of PALs as biocatalysts
for the amination of cinnamic acid analogues has been
described extensively in literature and implemented in
industrial processes,[7] the formation of d-amino acids has
not been reported. Furthermore to our knowledge the time-
dependence of the ee value has not been described previously.
To gain insight into the origin of d-amino acid synthesis,
the AvPAL-mediated amination of 4-nitrocinnamic acid (1m)
was studied in detail. The reactions were repeated with
purified PAL enzymes and confirmed that the variation in
ee values over time did not occur as a result of background
reactions taking place in the E. coli whole cells. Control
reactions in the absence of enzyme showed a) no conversion
into either the l- or d product and b) no racemization of
either l- or d-amino acids, thus eliminating the possibility of
non-selective background chemical reactions. The amination
of 1m was monitored over time using whole cell AvPAL
biocatalysts (15 mgmLÀ1). The reaction profile is consistent
with initial (reversible) formation of the l-amino acid as the
kinetic product followed by a slower process in which
formation of the d enantiomer occurs (Figure 2). The obser-
vation that the same equilibrium composition (1m: 14%, 2m:
86%, ee = À9%) was obtained starting from each of the three
components (i.e. 1m, l-2m, d-2m) under standard amination
reaction conditions supports this proposal.
The substrate range of eukaryotic PALs from the plant
Petroselinum crispum (PcPAL) and yeast Rhodotorula gluti-
nis (RgPAL) have been well characterized and show activity
towards a range of substituted phenylalanine derivatives. The
nature of the aromatic substituent has been shown to greatly
influence activity, providing insights into the enzymeꢀs
catalytic mechanism.[15] To date, there have been few reported
examples of bacterial PALs and they have not been previ-
ously exploited as biocatalysts for non-natural amino acid
synthesis. Recently, the crystal structure of a double mutant of
PAL from the bacteria Anabaena variabilis (AvPAL) was
solved, representing the first structure showing the flexible
catalytic loops well-resolved and in the active conformation
for catalysis.[12c] Availability of a detailed crystal structure
makes AvPAL an attractive candidate for directed evolution.
To investigate the suitability of AvPAL as a biocatalyst for
amino acid synthesis, the activity of wt AvPAL was deter-
mined alongside the eukaryotic PcPAL and RgPAL. The
conversion of a panel of cinnamic acid derivatives (1a–n;
(Table 1) into their corresponding amino acids were moni-
tored over time using E. coli BL21(DE3) whole cells
expressing the individual PALs. The activity of AvPAL was
largely comparable to that of PcPAL and RgPAL, with all
three enzymes catalyzing the conversion of the substrates 1a–
n with high to moderate conversions. All reactions initially
proceeded with excellent enantioselectivity in favor of the l-
amino acids (see the Supporting Information). Interestingly, it
was observed that the ee values of the products 2b, 2d–f, and
Table 1: The conversion and ee values of the cinnamic acid derivatives
1a–n (5 mm) after 22 h.
AvPAL
PcPAL
RgPAL
Conv. ee [%]
Conv. ee [%]
Conv. ee [%]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
1j
1k
1l
1m
1n
49
59
59
76
91
62
53
61
80
77
86
78
79
50
>99
>99
>99
71
>99
>99
>99
76
58
75
60
75
94
72
50
49
87
89
87
84
86
50
>99
6
>99
96
0
14
>99
À8
10
55
71
61
75
93
76
51
62
85
89
86
83
85
48
>99
2
>99
93
13
7
>99
38
28
Figure 2. The amination reaction of 4-nitrocinnamic acid (1m) medi-
ated by E. coli BL21(DE3) cells (15 mgmLÀ1) expressing AvPAL in 5m
NH4OH pH 9.5, 308C.
>99
75
0
1
À9
À11
À14
92
7
7
À6
À4
>99
5
>99
The active-site loop contains an essential Tyr78 residue,
which is believed to be responsible for abstraction of the
substrateꢀs C3 benzylic proton.[12c] To assess the role of the
catalytic base Tyr78 in d-amino acid formation, the Y78F
variant of AvPAL was expressed and purified, and was shown
>99
Percentage conversion and product ee values (for the l-enantiomer)
were determined by HPLC using a chiral stationary phase. E. coli
BL21(DE3) whole cells (20 mgmLÀ1) expressing each PAL were incu-
bated with 5 mm substrate in 5m NH4OH pH 9.5 at 308C.
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to be correctly folded by circular dichroism. Y78F showed no
activity towards the deamination of either enantiomer of
phenylalanine (2a) or 4-nitrophenylalanine (2m) and it was
also inactive towards the amination of 1a and 1m. Our results
confirm that Tyr78 plays a vital role in the AvPAL-catalyzed
deamination of l-2a and also demonstrates that it is an
essential catalytic residue for activity towards production of
d-amino acids.
tion.[11a] Treatment of wt AvPAL with NaBH4 led to reduced
enzyme which displayed similar activity to that of S168A (see
the Supporting Information). A Y78F/S168A double mutant
was expressed and displayed no activity towards 1m, l-2m, or
d-2m, demonstrating that the Tyr78 residue is essential for the
cofactor independent reactions and that the non-selective
pathway occurs in the active site rather than an alternative
binding site.
To examine the role of the prosthetic group in d-amino
acid formation the AvPAL S168A variant was expressed,
which can no longer form MIO by post-translational modi-
fication. The absence of the cofactor in this variant was
confirmed by analysis of the UV difference spectra. The
discrete maxima at about l = 310 nm, associated with the
presence of cofactor in wt AvPAL, is diminished in the S168A
variant.[16] The kinetic parameters for S168A and wt AvPAL
were determined. Surprisingly, the kcat value for wt AvPAL-
catalyzed amination of 1m was 140 times greater than that for
1a (Table 2). The S168A mutation resulted in complete loss of
activity towards the amination of 1a and deamination of l-2a.
However, S168A retained low level activity towards the
amination of 1m and towards the deamination of l-2m,
although a significant reduction (293- and 31-fold, respec-
tively) in the kcat values was observed. Interestingly, the
kcat value for the d-2m deamination was not significantly
affected by the absence of the MIO cofactor.
It has been previously shown that PAL-mediated deam-
ination of l-phenylalanine proceeds in a stereoselective
manner with loss of the pro-S benzylic proton.[17] To inves-
tigate the stereoselectivity of d-amino acid formation and
deamination, deuterated 3 (> 99% deuterium incorporation)
and deuterated d-4 (99% ee 83% 2H incorporation) and d-5
(98% ee 83% 2H incorporation) were synthesized by
a chemoenzymatic strategy. Of particular interest was the
application of N-acetyl amino acid racemase (NAAAR
G291D/F323Y)[18] in combination with a commercially avail-
able d-acylase to invert the C2 stereocenter to the required
R configuration for the synthesis of d-5 (see the Supporting
Information).
Amination of 3 was carried out in the presence of AvPAL,
producing a mixture of l- and d-amino acids (ee = 6%) which
were isolated by ion-exchange chromatography. 1H NMR
analysis of this product shows the presence of a single
diastereoisomer resulting from anti addition of ammonia
across the double bond, as judged by comparison with
chemoenzymatically synthesized standards
(Figure 3). Single enantiomers (ee > 99%) of the deuterated
biotransformation products (l-4 and d-4) could be obtained
4
and
5
Table 2: Kinetic constants for the amination reactions of 1a and 1m and
the deamination reactions of 2a and 2m measured spectrophotometrically.
Substrate
wt AvPAL
KM cat/kM
[mm] [sÀ1 mmÀ1
S168A AvPAL
KM cat/kM
[mm] [sÀ1 mmÀ1
]
kcat
k
kcat
k
[sÀ1
]
]
[sÀ1
]
amination[a]
1a
0.029 0.039 0.744
4.106 0.720 5.703
n.d.
n.d
n.d
1m
0.014 0.062 0.225
deamination[b]
l-2a
0.491 0.298 1.648
n.d.
n.d.
n.d
n.d
n.d
n.d
d-2a
n.d
n.d
n.d
l-2m
d-2m
1.380 0.478 2.887
0.090 0.089 1.011
0.045 0.137 0.328
0.025 0.329 0.076
[a] Amination reaction in 5m NH4OH, pH 9.5, 308C. [b] Deamination
reactions in 0.1m buffer, pH 8.3, 308C. n.d=kinetic constants could not be
measured.
These results suggest that the dominant pathway for the
formation (or consumption) of l-amino acids is dependent on
the MIO cofactor, whereas the analogous reactions involving
the d enantiomers occur by a MIO-independent pathway.
Comparable kcat values are observed for the S168A-catalyzed
deamination of both l-2m and d-2m, demonstrating that the
cofactor-independent pathway proceeds in a non-stereoselec-
tive manner. Reduction of the MIO exocyclic methylene
group represents an alternative method of cofactor inactiva-
Figure 3. 1H NMR spectra of a) the unlabeled 4-nitro-d-phenylalanine
d-2 m, b, c) the deuterated 4-nitro-d-phenylalanine standards 4 and 5,
and d) the isolated product from the AvPAL-catalyzed amination of the
4-nitro-(3-2H)-cinnamic acid 3.
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by kinetic resolution using commercially available d-amino
acid oxidase from porcine kidney and l-amino acid oxidase
from Crotalus adamanteus.
established MIO-dependent pathway remains an essential
catalytic residue in the cofactor-independent pathway, dem-
onstrating that this enzyme-assisted reaction occurs within the
PAL active site. The deamination of d-amino acids proceeds
by a stepwise E1cB mechanism with initial deprotonation at
the benzylic position catalyzed by Tyr78. We propose that
electron-deficient substrates increase the stability of this
intermediate carbanion with respect to reprotonation, allow-
ing a slow MIO-independent amine elimination step to occur.
Our observations further suggest that cinnamic acid deriva-
tives are able to adopt two productive binding modes in the
PAL active site (Figure 4). Although two binding modes of
cinnamic acid have been reported to occur in the closely
related mutases, a second binding conformation has not been
described for lyases.[14,19a]
The stereoselectivity of AvPAL deamination reactions
was also investigated using the isotopically labeled substrates
d-4 and d-5. Although substrate 5 yielded the single fully
protiated product 1m, consistent with the exclusive anti eli-
mination of the 3R deuterium, substrate 4 gave a mixture of
1m and 3. The ratio of products produced is variable and
appears to be influenced by subtle changes in reaction
conditions. This result was somewhat unexpected considering
that the (reversible) amination of 4-nitro-(3-2H)-cinnamic
acid 3 was completely stereoselective to produce isomer 4.
PAL-catalyzed deamination reactions are performed at
pH 8.3 whereas reactions in the amine synthesis direction
are conducted at pH 9.5, which leads to a more
favorable equilibrium position. Deamination of 4
and 5 were repeated at pH 9.5 and in both cases
were shown to result in the formation of a single
product as a result of anti elimination of the
3R proton or deuterium, respectively. Deamina-
tion of 4 and 5 catalyzed by S168A (lacking the
MIO cofactor) showed a similar trend to wt
AvPAL. At both pH 8.3 and pH 9.5, 5 yielded
fully protiated 1m exclusively, whereas deamina-
tion of 4 gave a mixture of 1m and 3. The loss of
deuterium incorporation was significantly higher
at pH 8.3 than at pH 9.5.
These observations are consistent with the
deamination of d-amino acids proceeding by
a stepwise E1cB mechanism, with initial stereose-
lective deprotonation of the pro-R benzylic
proton catalyzed by Tyr78 and subsequent elim-
ination of ammonia. At pH 8.3 non-stereoselec-
tive reprotonation of the carbanion intermediate
from either bulk solvent or active-site residues
competes with amine elimination, resulting in
partial loss of the deuterium in the final product.
The rate of reprotonation is pH dependent and is
slower at pH 9.5 than at pH 8.3. This is consistent
with studies regarding the mechanism of amino
mutases CcTAM and TcPAM (from Taxus
plants), which report a loss in the product
deuterium incorporation and demonstrate that
Figure 4. Proposed MIO-dependent and independent pathways. EWG=electron-with-
proton exchange with bulk solvent is pH depen-
dent.[19] Docking l- and d-phenylalanine into the
AvPAL active site (see the Supporting Informa-
drawing group.
tion) demonstrates that only the pro-S benzylic proton of l-
phenylalanine and pro-R proton of d-phenylalanine interact
with the catalytic Tyr78 residue, and is consistent with the
results of the isotopic labeling study.
Combined, the kinetic data obtained with wt AvPAL and
selected variants along with isotopic-labeling studies and
molecular modelling have provided significant mechanistic
insights into a competing MIO-independent pathway. This
pathway proceeds in a nonselective manner, leading to the
formation of both l- and d-amino acids. Hydroamination has
been shown to occur by anti addition of the amine and the
benzylic proton. The key catalytic base (Tyr78) in the well-
In conclusion, amination reactions catalyzed by both
bacterial AvPAL and eukaryotic PcPAL and RgPAL led to
significant formation of d-amino acids in addition to the
expected l-amino acid products. For substrates possessing an
electron-deficient aromatic ring, a significant reduction in
ee value was observed over time. Mutagenesis of key active-
site residues and the determination of kinetic parameters has
shown that d-amino acids are formed by an enzyme-assisted
MIO-independent pathway. The mechanism of this pathway
has been explored and the results are consistent with
a stepwise E1cB elimination process. The discovery of this
alternative pathway may provide insights for future studies on
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Published online: April 1, 2014
Keywords: hydroamination · amino acids · enzymes ·
.
isotopic labeling · reaction mechanisms
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