L LOSCHMIDT
, LABORATORIES
PROTEIN ENGINEERING
10. EXAMPLE OF UTILIZING PROTEIN ENGINEERING TO ENHANCE
ENZYME STABILITY
Loschmidt Laboratories Department of Experimental Biology Masaryk University, Brno
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Outline
□ Process design criteria
□ Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel
- motivation
- aims
- results
- conclusions
□ Method of protein stabilization - patent application
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Process design criteria
□ higher activity at process conditions
□ increased process stability
□ increased thermostability to run at higher temperatures
□ stability to organic solvents
□ absence of substrate and/or product inhibition
□ increased selectivity (enantio-, regio-, chemo-)
□ accept new substrate
□ catalyse new reactions
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□ organic cosolvents can have a positive effect on catalysis
- improving substrate solubility
- alteration substrate specificity and enantioselectivity
- suppression of water-induced side reactions
□ higher concentration of organic co-solvents usually cause protein denaturation
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□ to identify mutations influencing stability of haloalkane dehalogenases in organic cosolvent
□ to construct haloalkane dehalogenase with improved stability in buffer containing DMSO
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Methods
□ error-prone PCR (epPCR) by Taq polymerase, MnCI2
□ screening by pH indicator assay in MTPs with 42 - 52% DMSO phenol red: red <—► yellow (pH lower than 6.6)
□ purification by affinity chromatography
□ thermodynamic stability and structural characterization
by circular dichroism, fluorescence spectroscopy, differential scanning calorimetry and X-ray crystallography
□ functional characterization and kinetic stability
by activity assay (Iwasaki method) and steady-state kinetics
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Methods
□ thermodynamic x kinetic stability
Definitions of various stability parameters.
Measure	Symbol	Type of stability	Definition
Free enerav of unfolding	AG,|	Thermodynamic	Chanae in Gibbs free enerav aoina from the folded to unfolded state
| Melting temperature	Tm	Thermodynamic	The temperature at which half of the protein is in the unfolded state
Unfolding equilibnum		i MemTcaynamic	1 ne concentration ot unroided species divided by tne concentration ot
constant			folded SDecies
| Half-concentration		Thermodynamic	The concentration of denaturant needed to unfold half of the protein
			{chemical equivalentTH^^
Observed deactivation rate		Kinetic	Overall rate constant for going from native to deactivation species
^nnslanL			
1 Half-life		Kinetic	Time required for residual activity to be reduced to half
Temperature of half-	Tso	Kinetic	Temperature of incubation to reduce residual activity by half during a
i n act i Vatican			defined time period
Optimum temperature	'"opt	Kinetic	Temperature leading to highest activity
Total turnover number	TTIM	Kinetic	Moles of product produced over the lifetime of the catalyst
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Methods
□ site-directed mutagenesis by QuikChange
□ gene synthesis
□ saturation mutagenesis by inverse PCR using a synthetic oligonucleotide with one degenerated NNK codon
□ molecular basis of resistance to organic cosolvent
by molecular dynamics simulations in 40% DMSO
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Studied HLD
DhaA
from Rhodococcus rhodochrous
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4 positive hits
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"8 -\-1-1-1-1-1—
180     195     210     225     240 255
Wavelength (nm)
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Mutant resistant to DMSO
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Mutant resistant to DMSO
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Mutant resistant to DMSO
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DhaA DhaA 57
56
melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h)
melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h)
melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h)
^ray, K.A. et al.: Adv. Synth. Catal. 343, 607-617 (2001)
Mutant resistant to DMSO
£ wt   57   60   61 63
DBE - 1,2 dibromoethane; IH - iodohexane
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Mutant resistant to DMSO
o
to
eř-CS
1.0 i
2 ~ 0.8 ^ 0.6 I J 0.4
'£! O
« |0.2
LI 3.
U
Q.
I/)
0.0
rli
wt   57   60   61 63
O
1.0 n
O
DhaA 63
DBE - 1,2 dibromoethane; IH - iodohexane
S -0.8
I I 0-4 H
'43 O
« |0.2 H
u
Q.
I/)
0.0
IH
i
wt   57   60   61 63
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melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h)
^ray, K.A. et al.: Adv. Synth. Catal. 343, 607-617 (2001)
Mutant resistant to DMSO
DhaA 85, DhaA 88
DBE - 1,2 dibromoethane; IH - iodohexane
it
3
O
I/)
eř-CS
>
u
<
500 i 400 300 200 100 0
DBE
wt   57   60   61   63   80   82   85 88
r- 300 i
*t 250
§ 200 ■
to
1 150 -
O
C >
(J <
100 50 0
IH
-Í-		H-1
	-5-	
wt   57   60   61   63   80   82   85 88
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Mutant resistant to DMSO
Q.
wt   57   60   61   63   80   82   85 88
DBE - 1,2 dibromoethane; IH - iodohexane
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melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h)
^ray, K.A. et al.: Adv. Synth. Catal. 343, 607-617 (2001)
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Mutant resistant to DMSO
DhaA wt DhaA 57 DhaA 80
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Conclusion
□ resistance towards organic cosolvents correlates with thermostability
□ mutations lining access tunnel modulate occupancy of active site by solvent and can stabilize protein
□ robust catalysts were developed:
4 point mutations, 7~m | 19 C, T1/2 (40% DMSO) min -> days
□ engineering of access tunnels represents novel strategy for engineering of robust catalysts
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Conclusion
Angewandte
Chemie
Protein Stability
DOI: 10.1002/anie.201206708
Engineering Enzyme Stability and Resistance to an Organic Cosolvent by Modification of Residues in the Access Tunnel**
Tana Koudelakova, Radka Chaloupková, Jan Brezovsky, Zbyněk Prokop, Eva Šebestová, Martin Hesseler, Mořte za Khabiri, Maryia Plevaka, Dary na Kulík, Ivana Kuta Smatanova, Pavlína Řezačova, Rudiger Ettrich, Uwe T. Bornscheuer, and Jiri Damborsky*
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Method of protein stabilization
□ method for modification of the access routes in order to achieve better stability of protein towards temperature and solvents
□ definition of the access routes: channel x tunnel
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Method of protein stabilization
□ method for modification of the access routes in order to achieve better stability of protein towards temperature and solvents
□ definition of the access routes: channel x tunnel
□ general concept, tunnels found in all enzyme classes
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1. OXIDOREDUKTASES 2. TRANSFERASES 3. HYDROLASES
Cytochrome CYP3A4 Chalcone synthase Acetylcholinesterase
EC 1.1.3.6 EC 2.3.1.74 EC 3.1.1.7
4. LYASES 5. ISOMERASES 6. LIGASES
Tryptophan synthase Methylmalonyl-CoA mutase Asparagine synthetase
EC 4.2.1.20 EC 5.4.99.2 EC 6.3.1.1
Method of protein stabilization
□ procedure of protein stabilization
- identification the amino acids lining access routes based on knowledge of structure (CAVER, HotSpot Wizard)
- modification of selected amino acids „hot spots" (site-directed mutagenesis, random mutagenesis)
- analysis of constructed variants/libraries, assessment of the result of modification
□ rational focused mutagenesis based on detailed knowledge of structure and function
- creation of small focused "smart" libraries
- increase likelihood of beneficially modifying property
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Method of protein stabilization
□ modification of shape and physico-chemical properties of tunnels
- selective discrimination between the molecules of a substrate/product and undesired solvent molecules inside the access routes
- strengthening of hydrophobic interactions within the tunnel
- thermostability enhancement
□ high thermostability and resistance against organic cosolvents = required process design criteria
□ invention describing the method of stabilization patented
Damborsky, 1, Prokop, Z., Koudelakova, T., Štěpánková, V., Chaloupková, R., Chovancova, E., Gora, A., Brezovský, 1, 2011: Method of thermostabilization of a protein and/or stabilization towards organic solvents. Patent PV 2011-680.
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Method of protein stabilization
□ identification of tunnels - CAVER1
www.caver.cz
chovancova E. eta/., 2012, PLoS Comp. Biol. 8: el002708
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Helpful references
□ Koudelakova, T. et al. (2013) Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel, Angew. Chem. Int. Ed. 52: 1959-1963
□ Gray, K.A. (2001) Rapid evolution of reversible denaturation and elevated melting in a microbial haloalkane dehalogenase, Adv. Synth. Catal. 343: 607-617
□ Chovancova, E. (2012) CAVER 3.0: A Tool for Analysis of Transport Pathways in Dynamic Protein Structures, PLoS Comput. Biol. 8: el002708
□ QUESTIONS?
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L LOSCHMIDT
, LABORATORIES
PROTEIN ENGINEERING
11. EXAMPLE OF UTILIZING PROTEIN ENGINEERING TO ENHANCE
ENZYME ENANTIOSELECTIVITY
Loschmidt Laboratories Department of Experimental Biology Masaryk University, Brno
36/36