Angewandte
Chemie
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.
Angew. Chem. Int. Ed. 2014, 53, 4652 –4656
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4653
Lovelock, Sarah L.
