Inducing high activity of a thermophilic enzyme at ambient temperatures by directed evolution

Article abstract of DOI:10.1039/c7cc05377k

The long-standing problem of achieving high activity of a thermophilic enzyme at low temperatures and short reaction times with little tradeoff in thermostability has been solved by directed evolution, an alcohol dehydrogenase found in hot springs serving as the catalyst in enantioselective ketone reductions.

Full text of DOI:10.1039/c7cc05377k

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Inducing High Activity of a Thermophilic Enzyme at Ambient
Temperatures by Directed Evolution
Guangyue Li a,b, Miguel A. MariaꢀSolano c, Adrian RomeroꢀRivera c, Sílvia Osuna * c and Manfred
T. Reetz * a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The long-standing problem of achieving high activity of a the melting temperature (Tm) increasing from 57 ºC to 71 ºC, and the
thermophilic enzyme at low temperatures and short reaction kcatꢀvalue decreasing from 720 sꢀ1 to 470 sꢀ1 at 30 ºC. 6 This kind of
times with little tradeoff in thermostability has been solved by approach has been reviewed.7 The mutational effects have been
directed evolution, an alcohol dehydrogenase found in hot traced to protein rigidification due to newly introduced
springs serving as the catalyst in enantioselective ketone intramolecular Hꢀbonds and salt bridges as well as disulfide bond
reductions.
formations. In the present approach the opposite effect can be
anticipated, namely increased flexibility especially around the active
site. Thus, a strategy complementary to the traditional approach
would be of theoretical and practical interest. As will be seen, our
results are also relevant to the current debate in evolutionary biology
regarding changes of enzyme activity and stability starting from a
hot environment to a cooled earth over a period of three billion
years.8
Robust enzymes derived from thermophilic organisms that thrive
under extreme conditions as in hot springs are valuable catalysts in
such processes as paper production, baking, laundry detergents and
wasteꢀtreatment which operate at elevated temperatures.1,2 At room
temperature these enzymes generally show no activity or low
turnover, which is inacceptable for other types of applications, as in
the production of chiral pharmaceuticals or other fine chemicals.3
For practical (industrial) applications, maximal stability and activity
are needed, yet these appear to be opposing properties. Combining
the virtues of pronounced enzyme robustness with high activity at
ambient temperatures would lower energy expenditure and enable
shorter reaction times under operating conditions, enabling high
spaceꢀtime yields.3,4 A limited number of protein engineering studies
of such thermostable enzymes using rational design or directed
evolution based on mutator strains, epPCR and/or DNA shuffling
have appeared.5 The improvements proved to be moderate, generally
with a tradeoff in thermostability.
The present study likewise focuses on increasing activity of a
(hyper)thermally stable enzyme, but this time utilizing a different
directed evolution technique. Our goal is opposite to that of
conventional thermostabilization of mesophilic enzymes by directed
evolution, the usual alternative that is generally accompanied by a
tradeoff in activity. For example, Arnold et al. applied six cycles of
random mutagenesis and DNA shuffling to the pꢀnitrobenzyl
esterase from Bacillus subtilis in order to enhance thermostability,
As the model thermophilic enzyme we chose the NAD(P)Hꢀ and
Znꢀdependent
alcohol
dehydrogenase
TbSADH9,10
from
Thermoanaerobacter brockii, first discovered in the hot springs of
Yellowstone Park.9a It is identical to Thermoanaerobacter
ethanolicus (TeSADH) from a different source, a previously used
designation. In the purified form this ADH displays high
thermostability as demonstrated by a halfꢀlife of 1.7 hours at 90ºC
and 1.2 days at 80ºC.9f Thermostability as measured by differential
scanning microcalorimetry is Tm
=
98.5ºC.9e Using circular
dichroism (CD), we determined Tm to be 90ºC, which is in the range
of many hyperthermally stable enzymes.5 It has been noted that in
the reduction of a variety of structurally different ketones at ambient
temperatures using this enzyme long reaction times of several days
are needed,9b and ketoꢀesters require 72ºC for reasonable
conversion,11a as also reported for other thermophilic ADHs.11b In
other cases, overnight reactions had to be performed.9 In previous
studies, protein engineering of TbSADH was applied for various
purposes, including the increase and reversal of stereoselectivity for
different substrates,12 but a significant tradeoff in stability was often
noted12g,12h or thermostability was not measured.12f
The purpose of the present study was to evolve high activity of a
thermophilic enzyme at low temperatures, enabling short reaction
times for complete conversion while maintaining robustness. The
TbSADHꢀcatalyzed asymmetric reduction of acetophenone (1a) was
chosen as the model system with enantioselectivity playing a
secondary role (Scheme 1). Like many other thermostable ADHs,13
TbSADH shows very low activity towards 1a (and similar
substrates)9,11a at 30ºC, and requires extended reaction times and
more forcing conditions (e.g., overnight at 50ꢀ60ºC).9 The wildtype
(WT) is slightly (S)ꢀselective (17ꢀ18% ee).12h
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O
OH
R2
(S)-2a-d
OH
R2
(R)-2a-d
variants show, relative to WT, 58ꢀ and 52ꢀfold increases in kcat and
301ꢀ and 61ꢀfold improvements in catalytic efficiency (kcat/Km),
respectively. At 45ºC, variants TbSADHꢀ1 and TbSADHꢀ2 also
show notably better catalytic performance than WT, namely 51ꢀ and
36ꢀfold increases in kcat and improvements in kcat/Km by factors of
216 and 52ꢀfold, respectively.
The best mutants TbSADHꢀ1 and TbSADHꢀ2 were tested in
upscaled reactions at different temperatures using 50 mM of
substrate 1a in 1 mL of reaction volume (Table S4). Excellent results
were achieved, e.g., at 30ºC both variants ensured 96% conversion
within 1.5 hour with complete enantioselectivity (>99% ee (R)). In
contrast, at the same temperature WT TbSADH required 20 hours
for a mere 4% conversion and 17% ee (S). In further experiments,
whole cell catalysis at 30ºC using TbSADHꢀ1 and TbSADHꢀ2 was
successfully performed using substrate 1a at concentrations ranging
between 200 mM to 2 M (Table S5).
ADH
NAD(P)H
+
R1
1a-d
1a R1=Ph, R2=CH3
1b R1=Ph, R2=CH2CH3
1c R1=Ph(CH2)2, R2=CH3
1d R1=PhOCH2, R2=CH3
R2
R1
R1
Scheme 1. Asymmetric reduction of ketones 1aꢀd catalyzed by
TbSADH mutants.
We also measured the kinetics of WT TbSADH and variants
TbSADHꢀ1 and TbSADHꢀ2 using ketones 1b-d, revealing similar
activity increases (Table S6). Synthetically useful results were
achieved once more, e.g., in the case of 1b both variants reaching
96% conversion within one hour with 98% ee (R) (Table S7). At the
same temperature WT TbSADH led to less than 5% conversion after
20 hours with poor (S)ꢀ selectivity (27% ee).
The thermostability of both variants was measured by
determining the melting temperature (Tm) using circular dichroism.
Relative to WT TbSADH (Tm = 90ºC), the robustness of the two best
variants TbSADHꢀ1(Tm = 84ºC) and TbSADHꢀ2 (Tm = 87.5ºC) is
lowered by only 6ºC and 2.5ºC, respectively (Fig. S1 and Table S9).
We then performed molecular dynamics (MD) simulations on the
WT enzyme and the A85G/I86A variant, firstly for explaining the
origin of dramatically enhanced activity at ambient temperature, and
secondly to understand the reversed enantioselectivity (see SI for
Figure 1. TbSADH structure model showing docked acetophenone
(1a) as substrate (in purple) based on the crystal structure of
wildtype (1YKF),8a which served as a guide for choosing amino acid
positions for saturation mutagenesis (in pink).
Two crystal structures of TbSADH have been reported,10 one
containing NAD(P)H which is the catalytically active form
displaying an open binding pocket and a wide “entrance channel”.10a
We employed this structure (1YKF) in order to build a model for
docking substrate 1a into the binding pocket. In this way 13 residues
were identified for potential saturation mutagenesis, 10 surrounding
the substrate [C37, S39, A85, I86, L107, W110, T154, Y267, L294
and C295], and the rest occupying positions in the entrance channel
[I49, C283 and M285] (Fig. 1). These positions were then subjected
individually to NNKꢀbased saturation mutagenesis in which all 20
canonical amino acids are used as combinatorial building blocks,
requiring in each case the screening of ~96 transformants for 95%
library coverage.14
In the miniꢀlibraries generated by randomization at positions
85, 86, 110, 283, 285 and 294, several mutants were discovered
showing more than a 2ꢀfold activity improvement, namely A85G,
I86C, I86E, I86A, W110I, W110L, W110E, C283V, M285L,
M285V, L294T and L294V. The libraries created at the other seven
positions failed to harbor significantly improved variants (Table S1).
This information was then used as a basis for performing saturation
mutagenesis at a relatively large 6ꢀresidue randomization site
defined by the above hot spots. The use of NNK codon degeneracy
would require for 95% library coverage the screening of >109
transformants.14 As a practical alternative requiring only 1728
transformants, an appropriate reduced amino acid alphabet14, 15 was
designed individually for each one of the six residues (Table S2).
The choice of the respective building blocks was guided by the
amino acid substitutions that had shown positive effects in the initial
NNKꢀbased single libraries. This library harbored several distinctly
improved variants (Table S3), the best ones being TbSADHꢀ1
(A85G/I86A) and TbSADHꢀ2 (A85G/I86C) as shown by kinetic
experiments using purified proteins (Table 1). At 30ºC the two
computational details). In Figure
2 and S7, an overlay of
representative snapshots from the MD simulations performed in the
apo state is represented, together with the root mean square
fluctuations (RMSF) of all residues at 30 and 45ºC. The analysis of
RMSF allows us to identify the most flexible regions of the enzyme
structure, and rationalize the effect of the A85G/I86A mutations on
the TbSADH conformational dynamics. The loop composed of
residues 87ꢀ110 that partially covers the active site of the enzyme
(represented in yellow in Fig. 2AꢀB) is quite rigid in the case of the
WT enzyme. The introduction of A85G/I86A induces a higher
flexibility on the active site 87ꢀ110 loop, which is mainly due to a
change in the backbone conformation of residues 106ꢀ107 as
observed in the Ramachandran plot (see Figure S8, and SI movies).
Residues 106ꢀ107 are located close to the active site, and make
hydrophobic interactions with the substrate (see below). The change
in the backbone conformation of 106ꢀ107 increases the volume of
the active site (from ca. 96 Å3 for WT to ca. 117 Å3 for A85G/I86A,
(Fig. S9 and Table S12).
The higher flexibility of the A85G/I86A variant, especially in
the active site loop, confers the enzyme the ability to change the
shape of the active site easily and to adapt to the new nonꢀnatural
substrate, thus leading to higher activity at low temperatures.
We have also performed MD simulations in the presence of
acetophenone (1a) to elucidate the origin of reversed
enantioselectivity as done in a previous study (see SI for details).16
The higher flexibility of the active site loop 87ꢀ110 in the
A85G/I86A variant plays
a
key role in dictating the
enantioselectivity of the process. In WT, L107 occupies the small
binding pocket of the enzyme forcing 1a to position the phenyl
group in the large binding
2 | J. Name., 2012, 00, 1-3
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Table 1. Kinetic results using acetophenone (1a) as substrate.
Enzyme
Mutations
Km (mM)
kcat (min−1)
kcat/Km
(min−1 M−1)
30ºC
45ºC
WT TbSADH
TbSADHꢀ1
TbSADHꢀ2
WT TbSADH
TbSADHꢀ1
TbSADHꢀ2
19.01±1.68
3.70±0.17
16.20±2.46
20.78±1.25
4.95±0.25
14.47±2.05
1.80±0.07
94
A85G/I86A
A85G/I86C
104.76±1.31
93.15±2.35
3.79±0.15
28313
5750
182
A85G/I86A
A85G/I86A
194.54±3.14
136.04±9.32
39301
9402
Figure 2. Overlay of representative snapshots for WT (A) and A85G/I86A variant (B) in the apo state at 30°C. Average values of Root Mean
Square Fluctuation (RMSF) of all residues computed from the MD simulations in the apo state (C)
pocket. This orientation maximizes the CH··π and CH··CH the process, as it changes the preferred orientation of the substrate in
interactions of 1a and W110, L107, A85, and favors the proꢀ(S) the active site pocket.
pose. The higher flexibility of the active site loop in the A85G/I86A
Our findings have bearing on a recent study in which the
variant allows 1a to position the phenyl ring in the small binding putative evolutionary drivers of thermoadaptation in enzyme
pocket, thus favoring the formation of the (R)ꢀproduct. This proꢀ(R) catalysis were identified.8a On the basis of the hotꢀstart hypothesis of
orientation is stabilized by CH··π and CH··CH interactions between ancestral proteins,8 the authors note that “the challenge of evolving
1a and residues W110, A86, and L294 (see Fig. S10 and S11).
efficient enzymatic turnover at lower temperatures has not been
addressed”, emphasizing that the traditional concept of
stability/activity tradeoff needs to be questioned in Darwinian
evolution. While care must be taken when comparing natural with
laboratory evolution, our results demonstrate the physical feasibility
of evolving such mutational effects. On the practical side, the present
mutagenesis approach needs to be generalized by including other
(hyper)thermostable enzymes. It will be interesting to see if
flexibilization around the binding pocket is a general phenomenon
characteristic of such enzyme mutants.
Conclusions
In conclusion, we have applied an efficient directed evolution
strategy to evolve high activity of the thermophilic alcohol
dehydrogenase TbSADH at ambient temperatures with little tradeoff
in thermostability. Ketones such as acetophenone are rapidly
reduced with pronounced enantioselectivity (99% ee) at ambient
temperatures within short reaction times. The high thermostability of
the mutant(s) suggests that further mutational changes if needed for
other purposes can be tolerated.17 The respective molecular
phenomenon, uncovered by MD simulations, points to notably
enhanced flexibility of an active site loop. A comparison of the
movies of the wildtype and one of the mutants at the respective
binding pockets nicely visualizes the underlying effect. This confers
the active site pocket higher plasticity and the ability to adapt to new
nonꢀnatural substrates at lower temperatures. Higher flexibility of
the active site loop also has implications in the enantioselectivity of
Acknowledgements
We thank the MaxꢀPlanckꢀSociety and the LOEWE cluster
SynChemBio for generous support. A.R.R. thanks the Generalitat de
Catalunya for PhD fellowship (2015ꢀFIꢀBꢀ00165), M.A.M.S is
grateful to the Spanish MINECO for PhD fellowship (BESꢀ2015ꢀ
074964). S.O. thanks the Spanish MINECO for project CTQ2014ꢀ
59212ꢀP, Ramón y Cajal contract (RYCꢀ2014ꢀ16846), the European
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European Union’s Horizon 2020 research and innovation
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computer resources, technical expertise, and assistance provided by
the Barcelona Supercomputing Center
Supercomputacion.
ꢀ Centro Nacional de
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