1. Computational and Systems Biology
  2. Structural Biology and Molecular Biophysics
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Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering

  1. Friso S Aalbers
  2. Maximilian JLJ Fürst
  3. Stefano Rovida
  4. Milos Trajkovic
  5. J Rubén Gómez Castellanos
  6. Sebastian Bartsch
  7. Andreas Vogel
  8. Andrea Mattevi
  9. Marco W Fraaije  Is a corresponding author
  1. Molecular Enzymology Group, University of Groningen, Netherlands
  2. Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Italy
  3. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, United Kingdom
  4. c-LEcta GmbH, Germany
Research Article
Cite this article as: eLife 2020;9:e54639 doi: 10.7554/eLife.54639
7 figures, 6 tables and 5 additional files


The crystal structure of ADHA.

The figure highlights the final weighted 2Fo-Fc map for NADP+ bound to a subunit of the wild-type ADHA (subunit A, contour level 1.2 σ). The nicotinamide moiety of the cofactor is disordered and was not included in the final model.

Figure 2 with 1 supplement
Difference in Tm for 151 FRESCO-predicted ADHA mutants.

The average of two measurements is given and the standard error. The Tm of wild-type ADHA is 43 °C (set as 0). The 10 stabilizing mutations with a red bar were combined. Melting curves of wild type and the final mutant (M9*) are depicted in Figure 2—figure supplement 1.

Figure 2—figure supplement 1
Melting curves.

Apparent melting temperatures of wild type (in grey) and M9* variant. RFU = relative fluorescence units. M9* displays a first melting peak at 79 °C, second peak at 88 °C. One curve is shown from three technical replicates.

Michaelis-Menten plots for kinetics with NADP+.

(A) ADHA wild type (B) M9 mutant. Note that the X-axis scaling is different. The inset of B presents the same data with a different Y-axis scaling. Plots are fitted with Michaelis-Menten in GraphPad prism 6.07.

Structure of the M9 mutant of ADHA with mutated resides highlighted.

(A and B) quaternary structure of M9. The tetramer is organized such that the N-termini are on the outside (on the edge of the top-down view of A and B), whereas the C-termini all point inwards; which is where most and the most stabilizing mutations were found. (A) M9 structure with all atoms represented as balls. The four monomers are shaded in various colours, highlighting the particular clustering of the observed stabilizing mutations. (B) The structure as ribbon model, superimposing the mutant (blue ribbon, red spheres indicate mutated residue) and the wild type (cyan ribbon, yellow spheres). (C) Colour scheme as in B. The loop (196-214) that is dislocated as a result of the S197E mutation, compared to the structure of wild-type ADHA. The shift is accompanied by a flip of R18 into the NADP-binding pocket, likely due to an electrostatic attraction from the mutant glutamate. As a result, the cofactor (green carbons) is only bound in the wild type, while absent from the mutant structure.

Dimer interface with the V238L mutation (ΔTm = 7 ˚C).

(A and B) indicate the different monomers in the ADHA tetramer.

Michaelis-Menten plots for kinetics with NADP+.

ADHA wild type (grey, triangles) and M9* mutant (M9 with S197E reverted) (black, diamonds). Plots are fitted with the Michaelis-Menten equation in GraphPad prism 6.07.

Figure 7 with 1 supplement
Properties of wild-type and M9* ADHA.

(A) Temperature-activity profile using cyclohexanol as substrate. The dashed lines indicate the Tof the wild type (at 43 °C) and apparent melting temperatures of M9* (78.5 °C and 88 °C) (B) Enzyme activity monitored over time at 37 °C (buffer composition: 50 mM Tris-HCl pH 7.5). Figure 7—figure supplement 1 depicts the enzyme activity over time for 18 days.

Figure 7—figure supplement 1
Long-term stability.

Long-term stability at 37 °C of the wild-type protein (in grey, triangles) compared to M9* (in black, diamonds). Plot fitted with one-phase decay in GraphPad prism 6.07.


Table 1
Initial measurements of ADHA WT activity and selectivity with several ketones.
Substrate (concentration)kobs* (U/mg)Enantiomeric excess product (ee)
Ethyl acetoacetate (100 mM)4.1n.d.
Cyclohexanone (50 mM)3.2n.a.
Ethyl 4-chloro-3-oxobutanoate (COBA) (50 mM)0.3> 99% (R)
4-Chloroacetophenone (4-CAP) (50 mM)2.497.2% (S)
  1. *kobs values are averages based on 2–3 replicates and for each average the error was smaller than 5%.

Table 2
ADHA mutants with highest ΔTm and retained activity.

In parentheses the temperature of the first unfolding event (minor peak) is given. Michaelis-Menten kinetics for wild type and M9 are shown in Figure 3.

Single mutantskobs*
Tm app
Wild type0.643.0-
L150F0.557.25 ± 0.2514.0 ± 0.25
G149A0.452.5 ± 09.5 ± 0
M157K0.451.0 ± 08.0 ± 0
S197E0.549 ± 17.5 ± 1
V238L0.450.0 ± 07.0 ± 0
G159A1.248.5 ± 05.5 ± 0
N212R1.148.0 ± 05.0 ± 0
T194Vn.d.47.0 ± 04.0 ± 0
V193Ln.d.46.5 ± 03.5 ± 0
V217Pn.d.46.0 ± 03.0 ± 0
Combination mutantskobs*
Tm app
M2 (L150F + M157K)0.464.0 ± 021.0 ± 0
M3 (M2 + S197E)0.169.0 ± 026.0 ± 0
M4 (M3 + V238L)0.272 (62) ± 0.2529.0 ± 0.25
M5 (M4 + N212R)0.275.5 (64) ± 032.5 ± 0
M6 (M5 + G149A)0.181.0 (69) ± 038.0 ± 0
M7 (M6 + G159A)0.185.0 (74) ± 042.0 ± 0
M8 (M7 + V193L + T194V)0.0590.0 (81) ± 0.2547.0 ± 0.25
M9 (M8 + V217P)0.0394.5 (84) ± 051.5 ± 0
M9* (M9 - S197E)0.888.0 (78.5) ± 045.0 ± 0
  1. *kobs values are averages based on 2–3 replicates and for each average the error was smaller than 5% (between ± 0.0015–0.04 U/mg). Cyclohexanol was used as substrate.

Table 3
Characteristics of wild-type, M9, and M9* ADHA.

Activity measurements were performed at 25 °C in duplicate or triplicate and the respective Michaelis-Menten plots are depicted in Figure 6. Melting curves are depicted in Figure 2—figure supplement 1. Conversions were performed with 5 µM of ADHA and 10 mM of prochiral ketone substrate: ethyl 4-chloro-3-oxobutanoate (COBA) and 4-chloroacetophenone (4-CAP). Details and chromatograms are provided in Supplementary file 4.

(s−1 mM−1)
Conversion and ee (COBA)Conversion and ee (4-CAP)
ADHA WT43.0 ± 01.1 ± 0.049.5 ± 1.2116 ± 35> 99%
> 99% ee (R)
> 99%
97.2% ee (S)
M994.5 ± 00.27 ± 0.021040 ± 1080.26 ± 0.2n.d.n.d.
M9*88.0 ± 00.7 ± 0.019.6 ± 0.773 ± 14.5> 99%
> 99% ee (R)
> 99%
98% ee (S)
Table 4
Final melting temperatures (Tm) of M9* in various cosolvents (20% v/v).
CosolventTm (°C)ΔTm (°C)
-88.0 ± 0-
Methanol76.0 ± 0.5−12
Ethanol71.5 ± 0.5−16.5
Isopropanol67.0 ± 0.5−21
Table 5
Studies that have applied FRESCO for stabilization of enzymes, to date.
Quaternary structureΔTmReference
Limonene epoxide hydrolaseLEH149Dimer+35 °C(Wijma et al., 2014)
Haloalkane dehalogenaseLinB250Monomer+22 °C(Floor et al., 2014)
Hydroxymethyl furfural oxidaseHMFO525Monomer+12 °C(Martin et al., 2018)
Peptide amidasePAM508Monomer+23 °C(Wu et al., 2016)
Halohydrin dehalogenaseHheC254Tetramer+28 °C(Arabnejad et al., 2017)
Cyclohexanone monooxygenaseCHMO529Monomer+13 °C(Fürst et al., 2019)
Glucose oxidaseGOX605Dimer+8.5 °C(Mu et al., 2019)
ω-Transaminaseω-TA455Dimer+23 °C(Meng et al., 2020)
Short-chain dehydrogenaseADHA246Tetramer+45 °CThis work
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)NEB 10-beta chemically competent E. coliNew England Biolabshttps://www.neb.com/C3019I
Sequence based reagentpBAD Golden gate vector (N-terminal 6xHis, araC, bla)This studyMolecular Enzymology Group, University of Groningen
PolymerasePfuUltra II Hotstart PCR Master MixAgilent Technologies https://www.agilent.com/600852
Commercial assay or kitNi chromatography resinGE Healthcare Life Sciences https://www.gelifesciences.com/
Software, algorithmRosettaRosetta Commons https://www.rosettacommons.org/RRID:SCR_015701
Software, algorithmFoldXFoldXhttp://foldxsuite.crg.eu/RRID:SCR_008522
Software, algorithmYASARAYASARA Biosciences GmbHhttp://www.yasara.org/RRID:SCR_017591
Software, algorithmFRESCO scriptshttps://groups.google.com/forum/#!forum/fresco-stabilization-of-proteins
Software, algorithmGraphPad PrismGraphPad Prism https://graphpad.comRRID:SCR_015807Version 6

Additional files

Supplementary file 1

Crystallographic data and activity-impairing mutations.

(A) Crystallographic data. (B) Table of stabilizing mutations that disrupt activity.

Supplementary file 2

SDS-PAGE analysis.

(A) Expression of ADHA at different temperatures. (B) Purification of SUMO-M9*.

Supplementary file 3

Correlation of predictions with the measured data.

(A) Correlation of predicted values and experimental melting point data. (B) Graph of correlation.

Supplementary file 4

Chromatograms of conversions.

(A) Settings for GC and HPLC. (B) Chromatograms GC. (C) Chromatograms HPLC.

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