Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

Article abstract of DOI:10.1002/anie.201813499

The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine-tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (k_cat) of the designer enzyme by almost 100-fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200-fold.

Full text of DOI:10.1002/anie.201813499

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Accepted Article
Title: Directed evolution of a designer enzyme featuring an unnatural
catalytic amino acid
Authors: Clemens Mayer, Christopher Dulson, Eswar Reddem, Andy-
Mark Thunnissen, and Gerard Roelfes
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201813499
Angew. Chem. 10.1002/ange.201813499
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Directed evolution of a designer enzyme featuring an unnatural
catalytic amino acid
Clemens Mayer[a]*, Christopher Dulson[a], Eswar Reddem[a], Andy-Mark W.H. Thunnissen[b] and Gerard
Roelfes[a]*
Abstract: The impressive rate accelerations that enzymes display in
nature often result from boosting the inherent catalytic activities of
side chains by their precise positioning inside a protein binding pocket.
Here we show that such fine-tuning is also possible for catalytic
unnatural amino acids. Specifically, we report the directed evolution
of a recently described designer enzyme, which utilizes an aniline side
chain to promote a model hydrazone formation reaction. Consecutive
rounds of directed evolution identified a number of mutations in the
promiscuous binding pocket, in which the unnatural amino acid is
embedded in the starting catalyst. When combined, these mutations
boost the turnover frequency (kcat) of the designer enzyme by almost
100-fold. Crucially, these gains result from strengthening the catalytic
contribution of the unnatural amino acid, as the engineered designer
enzymes outperform variants, in which the aniline side chain is
replaced with a catalytically inactive tyrosine residue, by >200-fold.
The enviable rates and selectivities with which enzymes catalyze
their transformations in nature have fueled efforts to create
designer enzymes that can promote new-to-nature reactions with
comparable proficiencies.[1–3] Toward this goal, several
approaches for enzyme design have been developed over the
Figure 1. A: Chemical structure of p-aminophenylalanine. B: Formation of an
iminium ion intermediate in presence of anilines accelerates hydrazone (X =
NH) and oxime (X = O) formation reactions (for clarity, the reversibility of these
past decades; those include de novo design,[4,5] (computationally-
aided) protein redesign,[6,7] or recruitment of (un)natural cofactors
reactions is not shown). C: Crystal structure of the LmrR_pAF homodimer (PDB:
to appropriate binding pockets.[8,9] Irrespective of the approach
6I8N). Catalytic aniline side chains (red) and Trp96 (pink) are shown as sticks.
The positions of the β-carbons of 13 additional residues that line the binding
pocket of LmrR_pAF are shown as spheres. A 3-(N-morpholino)propanesulfonic
though, the catalytic activities of the resulting designer enzymes
pale in comparison to those found in nature.[2] However, one
acid (MOPS) buffer molecule that was found to be sandwiched between the
important prospect of installing abiotic activities into
central tryptophans is omitted for clarity (see Supporting Information)
proteinaceous scaffolds is the ability to boost low starting activities
by mimicking the Darwinian algorithm in the laboratory.[10] Indeed,
the iterative cycle of (1) introducing diversity through mutations,
install uniquely reactive functionalities to promote their target
reactions (i.e. formylglycine in type-I-sulfatases).[18–20] Designer
enzymes that make use of catalytic unnatural amino acids are
(2) identifying improved catalyst, and (3) amplifying more efficient
enzyme variants – collectively referred to as directed evolution[11]
– has given rise to engineered designer enzymes that display rate
also distinct from protein engineering efforts, in which genetic
accelerations akin to those found in nature.[12–14]
code expansion strategies[21,22] have been used to install non-
In this report, we demonstrate that directed evolution is also
standard side chains to improve/alter hydrophobic packing or
a means to boost the proficiency of a novel class of designer
enzymes, those that feature an unnatural amino acid as a catalytic
residue.[15–17] Such designer catalysts mimic natural enzymes that
substrate recognition.[23,24]
We recently reported the design and characterization of a
designer enzyme, which utilizes
a
uniquely reactive
employ posttranslational modifications of active site residues to
p-aminophenylalanine (pAF, Fig. 1A) residue to promote
abiological hydrazone and oxime formation reactions.[25] More
specifically, introducing pAF at position 15 in the multidrug
resistance regulator from Lactococcus lactis (LmrR), resulted in
LmrR_pAF (previously assigned as LmrR_V15pAF), which
promotes the condensation reactions of aldehydes with
hydrazines or hydroxylamines through the formation of an
iminium ion intermediate with the unnatural side chain (Fig.
1B).[26,27] While the designer enzyme outperformed aniline in
solution by a factor ~560, the catalytic contribution of the
unnatural side chain remained modest. In fact, an LmrR_pAF
variant, which features a structurally similar but catalytically-
[*] Dr. C. Mayer, C. Dulson, Dr. E. Reddem, Prof. G. Roelfes
Stratingh Institute for Chemistry
University of Groningen
Nijenborgh 4, 9474 AG Groningen (The Netherlands)
E-mail: c.mayer@rug.nl, j.g.roelfes@rug.nl
Dr. A. M.W.H. Thunnissen
Groningen Biomolecular Sciences and Biotechnology Institute
University of Groningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under: https://doi.org/XXXX
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Figure 1. A: Reaction conditions for the model hydrazone formation between NBD-H and 4-HBA in cleared lysates. B: Close-up of the hydrophobic pore in
LmrR_pAF. Trp96s and pAF15s shown as sticks; β-carbons of positions that gave rise to improved variants in round 1 and 2 are shown as spheres (color code as
indicated). C: Evolutionary optimization of LmrR_pAF. Apparent catalytic efficiencies (kcat/Km)app) of selected variants and their improvement with respect to
LmrR_pAF (see Table S1 and Fig S3 for a more detailed analysis); errors are standard deviations of at least 3 independent experiments; color code as in Fig. 2B.
D: Comparison of saturation kinetics at a 4-HBA concentration of 5 mM for LmrR_pAF and the best variants obtained after two rounds of directed evolution. Errors
are standard deviations of at least 3 independent experiments.
inactive tyrosine residue instead of pAF (LmrR_Y), was only about
10-times less efficient the parent designer enzyme. Given the
power of the evolutionary algorithm, we surmised that the role
pAF for catalysis could be optimized by identifying beneficial
mutations of residues surrounding the unnatural side chain.
Since our initial report of LmrR_pAF, we were able to
elucidate its structure (Fig. 1C, see Supporting Information for
details). As anticipated, the uniquely reactive aniline side chains
are embedded in the hydrophobic pore at the homodimer
interface of LmrR_pAF. Moreover, pAF residues flank the central
tryptophans (W96s), which are key for recruiting planar, aromatic
(substrate) molecules.[28] Upon inspection of this rudimentary
active site, we identified 13 additional residues that line the
hydrophobic pore and, as a result, are in proximity to the aniline
side chain (Fig. 1C). We anticipated that targeting these residues
by site-directed mutagenesis could give rise to more proficient
LmrR_pAF variants.
In order to rapidly identify beneficial mutations in the
LmrR_pAF binding pocket, we aimed to establish a medium-
throughput screening assay that would allow for the parallel
evaluation of LmrR_pAF variants. For this, we first optimized
production of LmrR_pAF and a catalytically-inactive control
protein (RamR)[29] in 96-well format and prepared cleared lysates.
Incorporation of pAF was achieved by first introducing p-
azidophenylalanine in response to a stop codon at position 15
(using the helper plasmid pEVOL-pAzF)[30] and subsequently
reducing the azido group with tris(2-carboxyethyl)phosphine in
cell lysates (see Supporting Information). To assess the catalytic
activity of the resulting LmrR_pAF in this complex mixture, we
took advantage of the chromogenic hydrazone formation reaction
between 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and
4-hydroxybenzaldehyde (4-HBA, Fig. 2A).[31] These two
substrates (final concentrations: 50 µM NBD-H and 5 mM 4-HBA)
were added to the cleared lysates and product formation was
followed at 472 nm in a 96-well plate reader for 2 hours. Indeed,
lysates containing LmrR_pAF displayed 30% higher product
formation rates than those containing RamR (Fig. S1). Moreover,
consistent with its lower activity, LmrR_Y (the variant containing
tyrosine instead of pAF) did not provide significant rate
acceleration with respect to RamR (Fig. S1). Taken together,
these results suggest that it should be feasible to accurately
assess the activity of LmrR_pAF variants in parallel.
To identify beneficial mutations, we initially targeted 8
residues (Fig S2), which are either in close proximity to pAF15 or
have previously been identified to improve catalytic parameters of
unrelated, LmrR-based designer enzymes.[32–34] Libraries
targeting each position were constructed using degenerate
primers (NNK codons, which allow for all 20 canonical amino
acids) and ~400 library members were evaluated following the
previously established screen. Two variants LmrR_pAF_A92R
and LmrR_pAF_L18K, which gave large improvements of 2.46 ±
0.13 and 2.04 ± 0.11 when compared to LmrR_pAF, were
subsequently produced and purified (Fig. S2, see Supporting
Information). Upon determining their catalytic parameters, both
variants displayed improved apparent catalytic efficiencies
((kcat/Km)app), when compared to the parent designer enzyme (2.2-
times for A92R and 1.5-times for L18K, Fig. 2A, Fig. S3 and
Table S1). Combined, these results attest that reproducible gains
in the screen translate into improved catalytic parameters for
purified LmrR_pAF variants.
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Table 1. Steady-state parameters of parent and engineered designer enzymes.
EM[a]
(M)
catalyst a]
kcat x 102 (s-1)
KNBD-H (µM)
K4-HBA (mM)
kcat /KNBD-H K4-HBA (M-2 s-1)
1 / KTS‡ (M-1)[b]
vs aniline[c]
LmrR_pAF
0.05 (0.002)
4.53 (0.33)
2.76 (0.11)
100 (7)
48 (4)
49 (2)
7.92 (0.49)
46.4 (4.3)
18.9 (1.0)
630 (60)
1.26
115
1.6 x 106
5.2 x 107
7.5 x 107
560
LmrR_pAF_RMH
LmrR_pAF_RMHL
20,500 (2,500)
29,500 (2,000)
18,400
26,500
69.8
Determined at 25 °C in phosphate buffer (50 mM) containing NaCl (150 mM) and 5% (v/v) DMF at pH 7.4. The estimated errors reflect the standard deviations
of at least three independent experiments. Under the same conditions kuncat = 3.95 x 10-4 M-1 s-1 and Kaniline = 1.12 M-2 s-1.[16] [a] effective molarity (EM = kcat/kuncat
)
‡
[c]
[b] chemical proficiency (1/KTS = [kcat/(KNBD-H · K4-HBA)]/kuncat vs aniline = ([kcat/(KNBD-H · K4-HBA)] / Kaniline) comparison of apparent third order rate constants of
designer enzymes and aniline.
Encouraged by these results, we chose LmrR_pAF_R
(containing the A92R mutation) as the template for a new round
of mutagenesis. In total, 12 additional positions were randomized
and 84 library members per targeted position screened (~1000
variants in total, Fig. S2). We identified another three mutations
(A11L, N19M, and F93H, but not L18K), which independently led
accelerate the bimolecular hydrazone formation. Instead, higher
catalytic efficiencies are the result of higher turnover frequencies
(kcat), which, again, is consistent with boosting the performance of
pAF for catalysis. Notably, under saturation conditions
LmrR_pAF_RMH and LmrR_pAF_RMHL display kcat values 91
and 55 times higher than the parent designer enzyme. Moreover,
to significant rate accelerations with respect to LmrR_pAF_R (Fig. the engineered variants outperform aniline in solution by more
2B-C). To identify potential synergistic effects between these
three mutations we constructed LmrR_pAF_R variants that
combined
two or all three of these mutations. Strikingly, the combination of
N19M and F93H (LmrR_pAF_RMH from here onward) provided
an exceptionally large improvement compared to either of the
other variants tested (Figs. 2C-D, Fig. S3 and Table S1). In fact,
when comparing the apparent catalytic efficiencies,
LmrR_pAF_RMH was 57-times more efficient than the parent
designer enzyme. The variant that featured the additional A11L
than 4 orders of magnitude (Table 1). Thus, while milimolar
concentrations of aniline are required to observe appreciable rate
acceleration in our model hydrazone formation reaction,[25]
engineered LmrR_pAF variants give rise to the same increases at
a concentration of <1 µM.
Notably, fine-tuning the inherent catalytic potential of the
aniline side chain proved also beneficial for hydrazone formations
with aldehydes different than 4-HBA. In fact, both
LmrR_pAF_RMH
and
LmrR_pAF_RMHL
significantly
accelerated hydrazone formation in presence of a total of 7
aldehydes (Fig. S5, see Supporting Information for details). A
minor specialization for 4-HBA (the screening substrate), in the
engineered variants is apparent, as the evolved variants
displayed a slight preference for this aldehyde, while LmrR_pAF
did not. Another notable difference between the parent and the
engineered designer enzymes was the ability of the latter to
significantly accelerate the hydrazone formation in presence of 2-
formylbenzoic acid; LmrR_pAF itself did not provide appreciable
levels of activity, under which all other aldehydes did. Thus, these
results indicate that the directed evolution of unnatural amino acid
containing designer enzymes can generate highly active as well
as versatile catalysts.
In absence of available structural information, it is difficult to
rationalize how the identified mutations boost the catalytic
potential of the aniline side chain. Both A11L and N19M are
located one helical turn away from pAF15 (Fig. 2B) and are likely
involved in positioning the unnatural side chain in a productive
conformation. A92R and F93H are located opposite of pAF15.
The introduction of a permanent positive charge by A92R is
somewhat puzzling, as it could negatively impact the population
of the crucial iminium ion (Fig. 1A), due to charge-charge
repulsion.[35] However, in the formation of this covalent, transient
intermediate, negative species are formed, which in turn could be
stabilized by the guanidinium side chain.[31,36] The F93H mutation,
in combination with N19M, appears critical for the boosts
mutation
(LmrR_pAF_RMHL)
provided
another
~30%
improvement and, thus, outperformed LmrR_pAF by 74-times
(Fig. 2C-D).
Higher efficiencies for these two LmrR_pAF variants result
predominantly from an increase in the apparent turnover
frequency (kcat,app, Table S1) and, point toward the desired fine-
tuning of the inherent catalytic activity of the unnatural amino acid.
Consistent with such a scenario, mutation of pAF15 to tyrosine in
LmrR_pAF_RMH and LmrR_pAF_RMHL proved crippling,
resulting in an efficiency loss of 99.7% for the former and 99.5%
for the latter. Moreover, when compared to the apparent catalytic
efficiency determined for LmrR_Y, the addition of the three or four
identified mutations only led to modest improvements of 2.0 and
3.9-times, respectively (Fig. S3 and Table S1). These results
further underscore that the identified mutations tailor the catalytic
contribution of pAF.
The effect of the beneficial mutations in LmrR_pAF_RMH
and LmrR_pAF_RMHL on the kinetics was studied in more detail.
By measuring the dependence of the reaction velocity on NBD-H
concentration (10-100 µM) at several fixed 4-HBA concentrations
(3-20 mM), we obtained steady-state kinetic parameters for both
variants (Table 1 and Fig. S4). Notably, the two engineered
designer enzymes have significantly higher Km values for 4-HBA
than LmrR_pAF, an intriguing result, as increasing the affinity for
this substrate could have provided a straightforward means to
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Acknowledgements
The authors thank Dr. I. Drienovská for helpful advice throughout the
project. This work was supported by the European Research Council (ERC
starting grant no. 280010) and the Netherlands Organisation for Scientific
Research (NWO, Vici grant 724.013.003, and Veni grant 722.017.007).
G.R. acknowledges support from the Ministry of Education Culture and
Science (Gravitation programme no. 024.001.035). C.M. acknowledges a
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Keywords: Directed evolution • Enzyme catalysis • Enzyme
Design • Organocatalysis • Hydrazones
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Entry for the Table of Contents
COMMUNICATION
Clemens Mayer*, Christopher Dulson,
Eswar Reddem, Andy-Mark W.H.
Thunnissen & Gerard Roelfes*
Page No. – Page No.
Directed evolution of a designer
enzyme featuring an unnatural
catalytic amino acid
The directed evolution of a designer enzyme featuring a uniquely reactive aniline side
chain as catalytic residue (in red) is reported. Multiple beneficial mutations were
identified (blue), which when combined increase the turnover frequency (kcat) of the
designer enzyme by >90 times. Engineered variants also outperform aniline by >4
orders of magnitude. Critically, this drastic increase is achieved by boosting the
inherent catalytic activity of the aniline side chain.
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