Szymanski et al.
JOCArticle
servation indicates that PAM exhibits ammonia lyase activ-
ity and encourages the exploration of the reverse lyase
reaction to synthesize R- and β-amino acids (2 and 3,
respectively) (Scheme 1). Recently, we have observed that
PAM may catalyze the addition of ammonia to substituted
(E)-cinnamic acids.32 Here we describe the factors that
govern the kinetic parameters and selectivity of PAM-cata-
lyzed reactions, demonstrating that a broad range of enan-
tiopure R- and β-amino acids can be produced and defining
the substrate scope of this novel biocatalytic system.
FIGURE 1. Initial stages of two proposed catalytic mechanisms of
PAM.
β-lactams,23 and dihydrouracils.24 Methods which are free of
the typical limitations connected with kinetic resolutions,
such as low efficiency and purification issues, are scarce and
rely on the use of aspartases25 and aminotransferases.26 The
use of an aminomutase for the partial biotransformation of
R-phenylalanine and its derivatives into their respective β-
isomers has recently been described.27
Results and Discussion
Phenylalanine aminomutase (PAM) from Taxus chinensis
was obtained as described in our previous communication32
with high purity (>95% as judged by SDS-PAGE with
Coomassie staining).
In order to determine the substrate scope of PAM, a series
of substituted cinnamic acids was prepared in very good
yields (usually >90%) by the Knoevenagel-Doebner pro-
cedure.33 The obtained library of compounds was tested in
the enzyme-catalyzed addition of ammonia to the activated
olefinic bond (Scheme 1). For every substrate, the kinetic
parameters of the reaction (kcat and KM) were determined in
order to estimate the structural features that influence the
affinity and catalytic rate. The ratio of the initial rates of
formation of the isomers 2 and 3 (R/β ratio, Scheme 1) was
also determined, aiming at understanding the factors that
govern the regioselectivity of the enzymatic reaction. Finally,
the enantiomeric excess of the products was determined.
Addition of ammonia to cinnamic acid (Table 1, entry 1)
yielded a 1:1 mixture of R- and β-phenylalanine, with ex-
cellent enantioselectivity (>99% ee for both isomers). For
compounds 1f (Table 1, entry 6) and 1g (Table 1, entry 7), no
detectable activity was observed. Substrates with fluoro,
chloro, bromo, and methyl substituents are accepted by
PAM (Table 1, entries 2-5), but the presence of any of these
substituents at the ortho position results in almost exclusive
formation of R-phenylalanine analogues. This phenomenon
is likely to be caused by steric shielding of the β-position. The
affinity of PAM toward these substrates seems to be influ-
enced by the lipophilicity of the substituent, as the lowest KM
value (corresponding to the highest affinity) was observed
for the bromo-substituted substrate, while for the compound
with the most hydrophilic substituent (fluoro) the affinity is
the lowest. This observation suggests the existence of hydro-
phobic side chains of amino acids in the active site of PAM
around the ortho positions of the substrate.
Phenylalanine aminomutase (PAM) from Taxus chinensis
is a recently discovered enzyme that catalyzes the conversion
of R-phenylalanine to β-phenylalanine in the biochemical
route leading to the side chain of Taxol.28 Unlike the
aminomutases that require external cofactors,29 the activity
of PAM depends on a protein-derived internal cofactor, 5-
methylene-3,5-dihydroimidazol-4-one (MIO, Figure 1).
Two different catalytic mechanisms have been proposed
for PAM-catalyzed amination (Figure 1), in which either the
aromatic ring (Figure 1, A)30 or the amino group (Figure 1,
B)31 of the R-amino acid adds to MIO. In both cases, the
elimination of ammonia from the MIO adduct is facilitated,
and (E)-cinnamic acid is the product of this transformation.
Recently published crystal structures of a related enzyme,
tyrosine aminomutase (TAM), with covalently bound inhi-
bitors present in the active site,31 together with our observa-
tions that PAM-catalyzed readdition of ammonia to
cinnamic acid in the second step of the PAM reaction is
MIO-dependent,32 support the hypothesis that the MIO-
adduct is formed by a reaction with the amine group of the
substrate (Figure 1, B).
It has been confirmed experimentally that both (E)-cin-
namic acid and β-phenylalanine are formed during the
PAM-catalyzed conversion of R-phenylalanine.32 This ob-
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The PAM-catalyzed addition of ammonia to meta-sub-
stituted cinnamic acids yields a mixture of R- and β-amino
acids. In case of halogen substituents (Table 2, entries 1-3)
the R isomer 2 is dominating, while the use of 3-methylcin-
namic acid (1k) as a substrate leads to preferential formation
of 3-methyl-β-phenylalanine (3k). A possible explanation of
this effect of the ring substituent on the regioselectivity is
provided in the section on para-substituted cinnamic acids
(vide infra). For 3-methoxycinnamic acid (Table 2, entry 5)
and 3-hydroxycinnamic acid (Table 2, entry 6) no detectable
enzymatic activity was observed. Due to the limited data
obtained it is difficult to draw conclusions on the factors
(33) See the experimental section in the Supporting Information for
details.
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