K. Fesko et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5987–5990
5989
what higher kcat and kcat/Km parameters than alrY265A,9 it was also
tested under the optimized conditions. It proved to be less effective
for the synthetic reactions, affording lower yields of the aldol prod-
uct than alrY265A (data not shown). It is possible that alrY265K is
less stable under the preparative conditions or that high concentra-
tions of benzaldehyde favor non-productive reactions with Lys265
at the active site, blocking the binding pocket.
The substrate scope of the alrY265A variant was subsequently
evaluated using a representative panel of aldehyde acceptors. As
shown in Table 2, the enzyme accommodates a range of benzalde-
hyde derivatives (Table 2, entries 1–7), but aliphatic aldehydes ap-
pear to be poor substrates (Table 2, entries 8 and 9). The observed
aldol products are formed with high stereoselectivity with respect
lated.27 Furthermore, since the related enzymes are members of
the same structural class of PLP proteins, it will presumably be pos-
sible to augment the activity of alrY265A, and perhaps its selectiv-
ity, either via additional site-directed mutagenesis or by subjecting
the enzyme to multiple rounds of directed evolution.28
The ability of the reengineered racemase to utilize alanine as a
donor in the synthetic aldol reactions is even more striking, as such
activity is unprecedented in other catalysts. It reflects the racemase
origins of alrY265A and highlights the advantage of an engineering
approach for accessing novel activities unavailable to naturally
occurring catalysts. b-Hydroxy-a,a-disubstituted-a-amino acids
are interesting as enzyme inhibitors29–31 and as conformational
modifiers of physiologically active peptides.32,33 As a consequence,
improved versions of the reengineered racemase could be of con-
siderable synthetic utility, affording these densely functionalized
molecules in a single step from simple starting materials, namely
alanine and an aldehyde.
to the configuration at C , affording exclusively
D-amino acids
a
(ee > 99%), and moderate to high selectivity at Cb (de, 40–97%).
The syn diastereomer is the dominant product in all cases.
As seen previously for threonine aldolases,12 the best substrates
are aromatic aldehydes bearing electron-withdrawing groups. The
highest conversion was obtained with 3-nitrobenzaldehyde, which
In summary, the Y265A mutant of alanine racemase is a viable
catalyst for the synthesis of b-hydroxy-a-amino acids. Although its
gave
When the nitro group is in the para position, both yield and diaste-
reoselectivity decrease somewhat: -4-nitro-b-phenylserine, a pre-
D
-3-nitro-b-phenylserine in 55% yield and 85% de (entry 3).
activity must still be optimized, its favorable stereochemical prop-
erties and unusual scope suggest promising opportunities for fu-
ture applications.
D
cursor of a chloramphenicol isomer,17,18 was obtained in only 36%
yield and 40% de (entry 4). 2-Nitrobenzaldehyde is an even poorer
substrate (entry 2), presumably for steric reasons. Benzaldehyde
derivatives with electron-donating amine or hydroxyl substituents
are weak electrophiles and generally give less than one percent
conversion (entries 5, 6). Judging from color changes in reaction
mixtures containing 2-hydroxy-, 3-hydroxy-, 4-hydroxy- and 3,4-
dihydroxybenzaldehyde (yellow to orange), oxidative side reac-
tions are a potential problem and may compete with the desired
aldol condensation. Nevertheless, reaction with piperonal (entry
7) appears promising, despite the low yield, since it provides a ster-
Acknowledgments
The financial support of the Schweizerischer National-fonds,
the ETH Zürich, and the Austrian Fonds zur Förderung der wissens-
chaftlichen Forschung (FWF, within the W901-B05 DK Molecular
Enzymology project) is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
eoselective route to
precursor of DOPS and noradrenaline isomers.19,20
Interestingly, alrY265A also accepts -alanine as a donor in the
D-3,4-methylenedioxyphenylserine (70% de), a
D
References and notes
aldol reactions. Such activity has not been seen with natural thre-
onine aldolases, but follows from the observation that the modified
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racemase is able to cleave D-a
-methyl-b-phenylserine.8 Although
the aldol reaction between alanine and benzaldehyde did not re-
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conditions (entry 10), the more reactive 3-nitrobenzaldehyde gave
a
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11).
Our results thus demonstrate that alrY265A can be used for the
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a
lower yields obtained with the reengineered racemase compared
with the natural enzymes presumably reflects its 100- to 1000-fold
lower specific activity. As a consequence, higher catalyst concen-
trations and longer reaction times are needed to achieve significant
conversions. Under these conditions, catalyst stability may be an
issue. Applications of natural aldolases have been optimized by
continuously removing the product from the reaction mixture.21
The unfavorable equilibrium has also been successfully shifted in
the desired direction by coupling the aldol reaction with an irre-
versible decarboxylation catalyzed by a highly diastereoselective
tyrosine decarboxylase.22 This bienzymatic approach not only re-
sulted in quantitative conversions but also improved Cb selectivity.
Similar strategies are likely to extend the utility of alrY265A.
The stereoselectivity of the reengineered racemase (ee > 99%,
de > 40%) is of particular note, since it is at least comparable to that
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of the best
icantly better than typical
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racemases and -threonine aldolases share a common evolutionary
heritage, whereas -threonine aldolases are evolutionarily unre-
D
-threonine aldolase in the literature,12,15,23 and signif-
-specific enzymes.12,13,15,22,24–26 These
L
D
24. Misono, H.; Maeda, H.; Tuda, K.; Ueshima, S.; Miyazaki, N.; Nagata, S. Appl.
Environ. Microbiol. 2005, 71, 4602.
L