A promiscuous de Novo retro-aldolase catalyzes asymmetric michael additions via Schiff base intermediates

Article abstract of DOI:10.1002/anie.201500217

Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Sp

Full text of DOI:10.1002/anie.201500217

Angewandte
Chemie
DOI: 10.1002/anie.201500217
Enzyme Catalysis
Hot Paper
A Promiscuous De Novo Retro-Aldolase Catalyzes Asymmetric
Michael Additions via Schiff Base Intermediates**
Xavier Garrabou, Tobias Beck, and Donald Hilvert*
Abstract: Recent advances in computational design have
enabled the development of primitive enzymes for a range of
mechanistically distinct reactions. Here we show that the
rudimentary active sites of these catalysts can give rise to
useful chemical promiscuity. Specifically, RA95.5-8, designed
and evolved as a retro-aldolase, also promotes asymmetric
Michael additions of carbanions to unsaturated ketones with
high rates and selectivities. The reactions proceed by amine
catalysis, as indicated by mutagenesis and X-ray data. The
inherent flexibility and tunability of this catalyst should make it
a versatile platform for further optimization and/or mecha-
nistic diversification by directed evolution.
simplicity, evolutionary optimization has sometimes resulted
in extensive active-site remodeling.[3b] We hypothesized that it
might also confer promiscuous activities that could be
enhanced and diversified by protein engineering.[7] In this
respect, such catalysts might complement simple amine-based
organocatalysts,[8] channeling reactive intermediates along
specific trajectories, facilitating greater stereocontrol, and
offering higher reaction rates. Our initial efforts focused on
À
C C bond formation through Michael reactions.
RA95.5-8, which efficiently cleaves (R)-4-hydroxy-4-(6-
methoxy-naphthalen-2-yl)butan-2-one [(R)-methodol, see
Scheme 1a], has a snug pocket for the methyl group adjacent
to the carbonyl moiety and a large hydrophobic binding site to
accommodate the aromatic substituent.[3b] A set of unsatu-
rated methyl ketones was therefore prepared and tested as
potential Michael acceptor substrates (Supporting Informa-
tion, Scheme S1).[9] The ketones were expected to react with
Lys83, the catalytic amine, to form a covalently bound
iminium intermediate, which could be attacked by appropri-
ate nucleophiles. Michael additions of 1a–1g with a range of
donor molecules (2a–2e) were performed in the presence of
RA95.5-8 and monitored by HPLC (see the Supporting
Information for details). Although most combinations
afforded only small amounts of the new products, the reaction
of 1a and ethyl 2-cyanoacetate (2a) was particularly rapid and
high-yielding (Scheme 1b). The resulting Michael adduct
(3a) was isolated and fully characterized.
C
reating enzymes with novel activities and specificities is
challenging.[1] Computational methods offer a potentially
general means of designing such molecules. Powerful algo-
rithms have been used to produce primitive enzymes for
a range of mechanistically diverse reactions, including non-
biological transformations.[2] Although the starting activities
are typically modest, directed evolution can afford significant
improvements in rate and selectivity.[3] As such catalysts rely
on rudimentary sets of functional groups, they might also be
good starting points for divergent evolution.
Retro-aldolases that exploit amine catalysis are the
mechanistically most complex enzymes designed to
date.[2b,3a,4] They utilize a reactive active-site lysine to promote
a multistep reaction sequence involving several enzyme-
bound Schiff base intermediates. Following laboratory evo-
lution, activities approaching those of natural class I aldolases
have been attained.[3b] Nevertheless, when compared to
natural aldolases,[5] the designed binding pockets of these
enzymes are relatively unsophisticated, consisting primarily
of a reactive amine in an apolar binding pocket.[3b,6] Given this
Analysis of 3a revealed high enantiocontrol at the site of
addition (3S/3R = 98:2), whereas the central carbon atom of
[*] Dr. X. Garrabou, Dr. T. Beck, Prof. Dr. D. Hilvert
Laboratory of Organic Chemistry, ETH Zꢀrich
8093 Zꢀrich (Switzerland)
E-mail: hilvert@org.chem.ethz.ch
Dr. X. Garrabou
Instituto de Quꢁmica Avanzada de CataluÇa–CSIC
Jordi Girona 18-26, 08034 Barcelona (Spain)
[**] We thank the Laboratory for High Pressure Experiments and the
Small Molecule Crystallography Center at the ETH Zꢀrich for
experimental assistance. The Swiss National Science Foundation
(SNSF) and the ETH Zꢀrich generously supported this study. X.G.
and T.B. respectively acknowledge the Spanish Ministerio de
Educaciꢂn, Cultura y Deporte and the Marie Curie Action within the
FP7-PEOPLE program (IEF-2011-299400) for postdoctoral fellow-
ships.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201500217.
Scheme 1. Retro-aldol and Michael reactions catalyzed by the computa-
tionally designed and evolved RA95 enzymes.
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
the donor moiety freely epimerized in water. The absolute
configuration of the enzymatically controlled chiral center
was elucidated by subjecting the Michael adduct to a Krapcho
decarboxylation and a subsequent reduction as previously
described,[10] followed by X-ray analysis of the hydrochloride
salt of the chiral cyclic amine (2R,4R)-5a (see Scheme 2 and
the Supporting Information).
acceptors that were poorly converted by RA95.5-8, such as
(E)-4-phenylbut-3-en-2-one (1b) and (E)-4-(6-methoxy-
naphthalen-2-yl)but-3-en-2-one (1c; see the Supporting
Information). These results underscore the potential of the
retro-aldolase for evolutionary optimization. Multiple rounds
of random mutagenesis and screening might be used to create
a higher-affinity binding site for the donor, improve the
reactivity of poorer nucleophiles, or achieve the stereocon-
trolled installation of additional chiral centers.
Direct insights into the catalytic mechanism were
obtained by crystallization of T53L/K210H RA95.5-8 in
complex with a stable derivative of the Michael product.
The enzyme was modified with (3R)-4a, and the resulting
Schiff base adduct was reduced with NaBH4 (Scheme 2). The
structure of the complex, which was determined to 1.20 ꢀ
resolution, shows the ligand covalently bound to Lys83 with
its aromatic substituent occupying the apolar binding site
designed to bind the naphthyl ring of methodol (Figure 1A).
These observations support the hypothesis that Lys83 func-
tions as an amine catalyst in the enzymatic transformation,
increasing the electrophilicity of the acceptor through Schiff
base formation. Replacement of Lys83 with methionine led to
a 100-fold decrease in activity (see Table S2), providing
further corroboration for this mechanism. Although the
precise disposition of the reactants at the active site is
unknown, the X-ray structure suggests that the covalently
bound Schiff base adopts an extended conformation that
exposes the Re face of the olefin to attack at C4 by the
carbanion donor, which likely binds in a small, solvent-
accessible pocket formed between the tips of the L1 and L6
loops.
Comparison of T53L/K210H RA95.5-8 with structurally
characterized intermediates in the evolution of RA95.5-8
highlights a number of salient differences in structural motifs
near the active site (Figure 1B), which underscore the
pronounced flexibility of the scaffold. Compared to the
previously determined RA95 structures,[3b] loops L1 (resi-
dues 51–64) and L6 (residues 180–190), which displayed
a range of conformations and high thermal displacement
parameters in the other variants, are now extensively
disordered. Most of L1, which contains the T53L mutation,
was not resolved, whereas two conformations could be
modeled for a few residues in L6. Although the residues in
direct contact with the ligand were not observed, the
occupancy of one of the L6 conformations correlates with
that of the covalently bound ligand, suggesting that the
Michael adduct induces (partial) structure in this floppy
segment. Loop L7 (residues 211–215), adjacent to the K210H
mutation but far from the bound ligand, also adopts
a completely different backbone conformation than in other
RA95 structures. Furthermore, the a-helix formed by resi-
dues 233–239, consistently structured in previous variants, is
distorted in T53L/K210H RA95.5-8. The marked conforma-
tional flexibility of this scaffold is likely to be a significant
factor in fostering mechanistic promiscuity.[12]
Scheme 2. Preparation of the cyclic amine (2R,4R)-5a and derivatiza-
tion of T53L/K210H RA95.5-8 with the decarboxylated Michael adduct
(3R)-4a.
To optimize this promiscuous activity, nine positions in the
active site were individually randomized, and improved
variants were identified by monitoring depletion of 1a at
350 nm in a spectrophotometric kinetic assay in multi-well
plates. Two mutations, T53L and K210H, enhanced the
catalytic efficiency by a factor of approximately three without
significantly compromising the enantioselectivity of the
reaction (3S/3R = 96:4). The steady-state kinetic parameters
for the Michael addition catalyzed by T53L/K210H RA95.5-8
were obtained by globally fitting the data to a random binding
mechanism. The intersecting lines in the double-reciprocal
plot (Figure S3) indicate independent binding of 1a and 2a to
the active site.[11] Notably, the turnover number of T53L/
K210H RA95.5-8 (kcat = 0.217 Æ 0.004 sÀ1) is comparable to
that of RA95.5-8 for the cleavage of (R)-methodol (kcat
=
0.36 sÀ1). This value corresponds to a 6300-fold rate accel-
eration over the spontaneous background reaction. The
KM value for unsaturated ketone 1a (322 Æ 12 mm) is similar
to that for (R)-methodol with RA95.5-8 (230 mm), suggesting
an analogous binding mode, whereas the high KM value for
donor 2a (16.5 Æ 0.5 mm) may be attributed to the lack of an
explicitly evolved binding site. The steady-state parameters
obtained with a variant containing only the K210H mutation
(Tables S2 and S3, Figure S4) indicate that this substitution
primarily lowers the apparent KM value for the Michael
acceptor, whereas the T53L mutation is responsible for the
observed increase in kcat. Residue 53 is in close proximity to
Lys83 and may favorably perturb the pKa value of the
catalytic residue.
The ability of the artificial RA95.5-8 retro-aldolase to
activate an unsaturated ketone as an iminium ion enables
efficient catalysis of asymmetric Michael additions. This
activation mode is unique among the few reported examples
The improved synthetic utility of the mutant manifested
itself in the enantioselective Michael addition of 2a to
2
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
a carbonyl group, is broad in scope but mechanistically simple,
these catalysts could conceivably accelerate many of the
carbon–carbon bond-forming reactions promoted by organo-
catalysts. Moreover, such promiscuity could be quite general.
Although computational methods are not yet able to create
the precisely tailored active sites of highly evolved natural
enzymes, the construction of binding pockets containing
reactive acids, bases, and nucleophiles appears to be relatively
straightforward.[2b,c,16] It is likely that the promiscuous activ-
ities of these proteins can be amplified and refined by directed
evolution to generate diversified families of artificial enzymes
with broadened substrate and reaction scope.
Received: January 9, 2015
Published online: && &&, &&&&
Keywords: asymmetric catalysis · biocatalysis ·
.
directed evolution · enzyme design · enzyme promiscuity
[1] a) H. Kries, R. Blomberg, D. Hilvert, Curr. Opin. Chem. Biol.
2013, 17, 221 – 228; b) G. Kiss, N. C¸ elebi-ꢂlÅꢃm, R. Moretti, D.
Baker, K. N. Houk, Angew. Chem. Int. Ed. 2013, 52, 5700 – 5725;
Angew. Chem. 2013, 125, 5810 – 5836; c) D. Hilvert, Annu. Rev.
Biochem. 2013, 82, 447 – 470.
[2] a) D. N. Bolon, S. L. Mayo, Proc. Natl. Acad. Sci. USA 2001, 98,
14274 – 14279; b) L. Jiang, E. A. Althoff, F. R. Clemente, L.
Doyle, D. Rçthlisberger, A. Zanghellini, J. L. Gallaher, J. L.
Betker, F. Tanaka, C. F. Barbas III, D. Hilvert, K. N. Houk, B. L.
Stoddard, D. Baker, Science 2008, 319, 1387 – 1391; c) D.
Rçthlisberger, O. Khersonsky, A. M. Wollacott, L. Jiang, J.
DeChancie, J. Betker, J. L. Gallaher, E. A. Althoff, A. Zanghel-
lini, O. Dym, S. Albeck, K. N. Houk, D. S. Tawfik, D. Baker,
Nature 2008, 453, 190 – 195; d) J. B. Siegel, A. Zanghellini, H. M.
Lovick, G. Kiss, A. R. Lambert, J. L. St. Clair, J. L. Gallaher, D.
Hilvert, M. H. Gelb, B. L. Stoddard, K. N. Houk, F. E. Michael,
D. Baker, Science 2010, 329, 309 – 313; e) F. Richter, R. Blom-
berg, S. D. Khare, G. Kiss, A. P. Kuzin, A. J. T. Smith, J. Gallaher,
Z. Pianowski, R. C. Helgeson, A. Grjasnow, R. Xiao, J.
Seetharaman, M. Su, S. Vorobiev, S. Lew, F. Forouhar, G. J.
Kornhaber, J. F. Hunt, G. T. Montelione, L. Tong, K. N. Houk, D.
Hilvert, D. Baker, J. Am. Chem. Soc. 2012, 134, 16197 – 16206.
[3] a) E. A. Althoff, L. Wang, L. Jiang, L. Giger, J. K. Lassila, Z.
Wang, M. Smith, S. Hari, P. Kast, D. Herschlag, D. Hilvert, D.
Baker, Protein Sci. 2012, 21, 717 – 726; b) L. Giger, S. Caner, R.
Obexer, P. Kast, D. Baker, N. Ban, D. Hilvert, Nat. Chem. Biol.
2013, 9, 494 – 498; c) O. Khersonsky, G. Kiss, D. Rçthlisberger, O.
Dym, S. Albeck, K. N. Houk, D. Baker, D. S. Tawfik, Proc. Natl.
Acad. Sci. USA 2012, 109, 10358 – 10363; d) R. Blomberg, H.
Kries, D. M. Pinkas, P. R. E. Mittl, M. G. Grꢃtter, H. K. Privett,
S. L. Mayo, D. Hilvert, Nature 2013, 503, 418 – 421; e) N.
Preiswerk, T. Beck, J. D. Schulz, P. Milovnꢄc, C. Mayer, J. B.
Siegel, D. Baker, D. Hilvert, Proc. Natl. Acad. Sci. USA 2014,
111, 8013 – 8018.
Figure 1. A) Crystal structure of T53L/K210H RA95.5-8 and the Michael
adduct derivative (4R)-4a (in green) after chemical reduction of the
adduct covalently bound to Lys83. Positions subjected to random-
ization are highlighted in yellow, and the mutations introduced in this
work are depicted in orange. B) Alignment of T53L/K210H RA95.5-8
(orange, the mutated positions 53 and 210 are highlighted as spheres)
derivatized with (4R)-4a (green) and the previously solved structures
of RA95.0 (pale gray), RA95.5 (cyan), and RA95.5-5 (blue) derivatized
with a diketone analogue of methodol (pink). For each structure, the
distribution of the thermal displacement parameters within the model
is represented by the tube diameter.
of Michael reactions catalyzed by promiscuous enzymes,
which either increase the reactivity of the Michael acceptor
through hydrogen-bonding interactions with the oxyanion
hole of lipases[13] or activate aldehyde-based Michael donors
by enamine formation.[14] Although high catalytic rates[13a]
and enantioselectivities have been reported for these sys-
[4] S. Bjelic, Y. Kipnis, L. Wang, Z. Pianowski, S. Vorobiev, M. Su, J.
Seetharaman, R. Xiao, G. Kornhaber, J. F. Hunt, L. Tong, D.
Hilvert, D. Baker, J. Mol. Biol. 2014, 426, 256 – 271.
[5] T. Gefflaut, C. Blonski, J. Perie, M. Willson, Prog. Biophys. Mol.
Biol. 1995, 63, 301 – 340.
[6] a) J. K. Lassila, D. Baker, D. Herschlag, Proc. Natl. Acad. Sci.
USA 2010, 107, 4937 – 4942; b) L. Wang, E. A. Althoff, J. Bolduc,
L. Jiang, J. Moody, J. K. Lassila, L. Giger, D. Hilvert, B.
Stoddard, D. Baker, J. Mol. Biol. 2012, 415, 615 – 625; c) R.
Obexer, S. Studer, L. Giger, D. M. Pinkas, M. G. Grꢃtter, D.
Baker, D. Hilvert, ChemCatChem 2014, 6, 1043 – 1050.
tems,[14b,15] the combination of catalytic proficiency ([kcat
/
(KM,acceptor ꢁ KM,donor)]/kuncat = 1.2 ꢁ 109 mÀ1) and stereochemical
control achieved by T53L/K210H RA95.5-8 is remarkable for
an artificial enzyme.
In summary, our results indicate that computationally
designed retro-aldolases are a potentially valuable source of
promiscuous catalytic activity. As the chemistry of iminium
ions and enamines, formed through attack of an amine on
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
[7] a) M. D. Toscano, K. J. Woycechowsky, D. Hilvert, Angew.
Chem. Int. Ed. 2007, 46, 3212 – 3236; Angew. Chem. 2007, 119,
3274 – 3300; b) P. A. Romero, F. H. Arnold, Nat. Rev. Mol. Cell
Biol. 2009, 10, 866 – 876.
[8] a) A. Erkkilꢅ, I. Majander, P. M. Pihko, Chem. Rev. 2007, 107,
5416 – 5470; b) S. Mukherjee, J. W. Yang, S. Hoffmann, B. List,
Chem. Rev. 2007, 107, 5471 – 5569.
[14] a) E. Zandvoort, E. M. Geertsema, B.-J. Baas, W. J. Quax, G. J.
Poelarends, Angew. Chem. Int. Ed. 2012, 51, 1240 – 1243; Angew.
Chem. 2012, 124, 1266 – 1269; b) R. Weinstain, R. A. Lerner,
C. F. Barbas III, D. Shabat, J. Am. Chem. Soc. 2005, 127, 13104 –
13105; c) T. Kitazume, Z. Jiang, K. Kasai, Y. Mihara, M. Suzuki,
J. Fluorine Chem. 2003, 121, 205 – 212.
[15] E. M. Geertsema, Y. Miao, P. G. Tepper, P. Dehaan, E.
Zandvoort, G. J. Poelarends, Chem. Eur. J. 2013, 19, 14407 –
14410.
[9] K. Zumbansen, A. Dçhring, B. List, Adv. Synth. Catal. 2010, 352,
1135 – 1138.
[10] M. S. Taylor, D. N. Zalatan, A. M. Lerchner, E. N. Jacobsen, J.
Am. Chem. Soc. 2005, 127, 1313 – 1317.
[11] I. H. Segel, Enzyme kinetics: behavior and analysis of rapid
equilibrium and steady-state enzyme systems, Wiley, New York,
1993, p. 984.
[12] N. Tokuriki, D. S. Tawfik, Science 2009, 324, 203 – 207.
[13] a) M. Svedendahl, K. Hult, P. Berglund, J. Am. Chem. Soc. 2005,
127, 17988 – 17989; b) J.-F. Cai, Z. Guan, Y.-H. He, J. Mol. Catal.
B 2011, 68, 240 – 244.
[16] a) M. Merski, B. K. Shoichet, Proc. Natl. Acad. Sci. USA 2012,
109, 16179 – 16183; b) I. V. Korendovych, D. W. Kulp, Y. Wu, H.
Cheng, H. Roder, W. F. DeGrado, Proc. Natl. Acad. Sci. USA
2011, 108, 6823 – 6827; c) S. Rajagopalan, C. Wang, K. Yu, A. P.
Kuzin, F. Richter, S. Lew, A. E. Miklos, M. L. Matthews, J.
Seetharaman, M. Su, J. F. Hunt, B. F. Cravatt, D. Baker, Nat.
Chem. Biol. 2014, 10, 386 – 391.
4
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Enzyme Catalysis
Born promiscuous: Artificial enzymes
obtained by computational design and
directed evolution utilize relatively simple
catalytic machineries to achieve remark-
able levels of activity. These catalysts are
potentially a rich source of novel chemical
reactivity, as shown for the artificial retro-
aldolase RA95.5-8, which also efficiently
catalyzes asymmetric Michael additions
via iminium ion intermediates.
X. Garrabou, T. Beck,
D. Hilvert*
&&&& — &&&&
A Promiscuous De Novo Retro-Aldolase
Catalyzes Asymmetric Michael Additions
via Schiff Base Intermediates
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!