1. Directed and rationale evolution for production of new enzymes1

2. “Artificial” evolution
Definition:
System whereby the natural evolutionary process is mimicked in the laboratory under accelerated conditions to result in genes/gene products which possess novel, desired characteristics.
Why improve on natural evolution?
Biotechnology requires novel enzymes and proteins that nature will likely never evolve:
New catalysts for chemical synthesis (synthesis of enantiomers)
Additives to detergents (stability to pH, heat, bleach, solvents)
Plant resistance to insects and chemicals (synthetic toxins, herbicide resistance)
Novel proteins as therapeutics (antibodies, vaccines, gene therapy)

3. Generation of Diversity
Target
Protein
Activity
Stability
Selectivity
Activity in Solvents
Substrate Specificity
pH Profile
Cofactor Requirement
Generation of Diversity
1. Random Mutagenesis
2. Gene Recombination
Iterative Cycles
Relative Performance
Screen or Selection ……
Goal Achieved 1 2 3 4
Generations 3

4. Directed Evolution Field is Rapidly Expanding
pre-1990
Number of papers published
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004 4

5. What can be engineered in Proteins ? -> Folding (Structure):
Thermodynamic Stability
(Equilibrium between: Native  Unfolded state)
Environmental Stability
(Temperature, pH, Solvent, Detergents, Salts …..)
-> Function:
Binding
(Interaction of a protein with its surroundings
Catalysis
(Increased stability of the transition state  increased catalytic rates !!!) Requires: Knowledge of the Catalytic Mechanism !!!

6. Rational Protein Design Directed Evolution6

7. “You get what you screen for”7

8. Laccases: general features
Multi-copper-containing enzymes catalysing the oxidation of a wide spectrum of aromatic compounds, primarily phenols and anilines, along with reducing molecular oxygen to water.
The Cu1 is the primary electron acceptor site in laccase catalysed reaction. Four 1-electron oxidations of a reducing substrate occur at this site. The electron is then transferred, through the highly conserved His-Cys-His tripeptide, to the TNC, where O2 is reduced to water.

9. Laccases: origin and distribution
Laccase was first detected (1883) in the Japanese lac tree Toxicodendron verniciflua (formerly Rhus vernicifera).
Later, it was found in certain other plants, in many insects, and in a variety of fungi. It is particularly widespread in ligninolytic basidiomycetes, and more than 125 different basidiomycetous laccase genes have been described.
The occurrence of laccase in prokaryotes seems to be restricted to certain species.

10. Laccases from white-rot fungi
Glycoproteins (carbohydrate content between 10%-25%)
Acidic pI
Contain four copper atoms distributed into three redox sites: Type 1 (T1), Type 2 (T2) Type 3 (T3)
Monomeric structure of KDa
Laccase structure is organized in three domains each with -barrel type architecture
Many fungi produce several laccase isozymes differing with regard to optimum pH, substrate specificity, molecular weight, cellular localization, and quaternary structure. These enzymes are differentially expressed as function of the environmental growth conditions
Laccase gene families have been found in different basidiomycetes, indicating that they may have evolved through duplication-divergence events.
Domain 3 forms the cavity in which the type-1 copper is located
The tri-nuclear copper cluster (T2/T3) is embedded between domains 1 and 3 with both domains providing residues for the coordination of the coppers
Domain1 Domain2 Domain3

11. Laccases: origin and distribution
Due to their low substrate specifity laccases have widespread applications:
• Effluent decolourisation and detoxification
Pulp bleaching;
Textile industries;
• Biorefinery;
Conversion of chemical intermediates;
• Removal of phenols from wines;
• Fiber synthesis and grafting;
• Biosensors;
Biofuel cells;
Synthesis of drugs.

12. Laccases in Industry
Industrial enzyme market is valued at $2 billion per annum with a potential annual growth rate of 3 to 5%.
Laccase stake in this market is about 4% thus making it a potential $800 million market cap.
Source: BCC Research

13. CASE STUDY Laboratory evolution of laccases
There is no ideal laccase to fit for all purposes, but there exists a real possibility of designing improved industrial enzymes.
Directed evolution
Error-prone PCR
Family shuffling
Saturation mutagenesis
Low-energy ion implantation
Methane sulfonate-based tecnique
Chimeras
T1 Redox potential
Active site and substrate binding pocket
C-terminus role

14. CASE STUDY
Laboratory evolution of laccases 14

15. Understanding how the E° of copper sites in proteins is regulated and how E° and geometric and electronic structure perturbations influence the electron transfer function of a protein is one of the major challenges in the field of metallo-biochemistry.
Redox potentials exhibited by laccases span a broad range of values from 400 mV for plant laccases to 790 mV for some fungal laccases.
The conserved coordinating amino acids for the T1 copper site are two histidines and a cysteine, and the natural variations occur in the so called axial position with a single interaction from a methionine being the most common arrangement.
Fungal laccases have non-coordinating phenylalanine or leucine at this position and deep analyses have been undertaken to understand if this feature may contribute, at least in part, to the high E0 observed in these enzymes.

16. LEA segment  high E° VSG segment  low E°
In their pioneering work Xu and co-workers targeted a pentapeptide segment believed to be located near the T1 Cu site in laccase.
Based on sequence homology analysis, experiments of site-directed mutagenesis were planned. The existence of such a correlation was hypothesized:
LEA segment  high E°
VSG segment  low E°
Xu F, et al., (1998) Biochem J 334:63–70

17. Later, the resolution of Coprinus cinereus laccase structure, determined by X-ray crystallography, boosted rational choice of candidate residues for site-directed mutagenesis.
Mutagenesis experiments in Trametes villosa laccase helped to conceive that the lack of the fourth axial ligand in laccases is an important factor determining the higher values of E° displayed by laccases
Xu F, et al., (1999) J Biol Chem 274:

18. Over the last decade, the growing library of fungal laccase structures, determined by X-ray crystallography, along with spreading of in silico models has been an essential tool for structure-function relationships studies.
Site-directed mutagenesis has been used to replace T1 axial ligand Met502 in the CotA laccase from Bacillus subtilis by leucine or phenylalanine.
Integrated studies have been undertaken to compare wild-type and mutated forms of the CotA laccase.
Durão P, et al., (2006) J Biol Inorg Chem. 11:

19. M502L
M502F
X-ray structural comparison of M502L and M502F mutants with wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The slight movement of the mutated residue towards the protein surface, and away from the type 1copper atom, leads to a concerted movement of this region, pushing it away towards the solvent, and slightly increasing the exposure of the copper centre.
Thus, M502L and M502F mutants, display an increase of the redox potential by approximately 100 and 60 mV, respectively. This is most probably related to the stabilization of the reduced state of the copper atom, Cu(I), by the elimination of the axial ligand.
Furthermore, mutations in the axial ligand have a profound impact on the thermodynamic stability of the enzyme
Durão P, et al., (2006) J Biol Inorg Chem. 11:

20. Laboratory evolution of laccases
CASE STUDY
Laboratory evolution of laccases 20

21. When the first high resolution structure of a laccase (Trametes versicolor) with an organic reducing substrate, 2,5-xylidine in the substrate binding cavity was resolved, many hydrophobic protein–ligand interactions were shown to take place.
Residues His458 and Asp206 interact with the amino group of the reducing substrate
Bertrand T, et al., (2002) Biochemistry 41:7325–7333

22. Asp206 is well conserved among fungal laccases from basidiomycetes whereas glutamate can be found among ascomycetes.
New insights into the binding cavity of the reducing substrate of a Trametes versicolor laccase have been provided by site directed mutagenesis
Basidiomycetes
Ascomycetes
Plants
An AspAsn modification induced modifications in catalytic properties of the enzyme and led to a significant shift (DpH = 1.4) of the optimum towards higher pH which suggests significant alterations of the interactions between the reducing substrate and the binding pocket
Madzak C, et al., (2006) Protein Eng Des Sel 19:77–84

23. In the hetero-dimeric laccase POXA3 from Pleurotus ostreatus laccase, the well conserved Asp involved in substrate interaction is substituted with an Arg residue. Site directed mutants were produced in order to understand the role of this molecular determinant in determining peculiar properties of P. ostreatus laccases.
S264
D205
H466
W465 Cu
POXA1b
D210 Cu
H456
W455
L275
POXC
R211 Cu
H458
F345
Q340
N272
POXA3
A significant worsening of catalytic properties was observed along with a decrease of stability when Asp is removed from the substrate binding pocket
Autore F, et al., (2009) Enzyme Microb Technol 45:507–513 23

24. CASE STUDY SITE-DIRECTED MUTAGENESIS
Searching for POXA1b mutants to substitute the classical precursors of dye synthesis with cheaper ones
Cathecol , 5 Diaminobenzensulfonic acid
(2,5 DABSA) n +
Laccase + O2
Resorcinol , 4 Diaminobenzensulfonic acid
(2,4 DABSA) +
Laccase + O2

25. CASE STUDY
Development of POXA1b homology model. Homology modelling is possible only if the proteins have a percentage of identity ≥ 40% with the other proteins with known structures
Refinement of POXA1b homology model through Molecular Dynamics simulation.
MD simulation of 200 ns

26. CASE STUDY
In order to understand the lack of activity towards 2,4 DABSA two approaches were chosen:
calculation of ΔG through Quantuum Mechanics calculation
analysis of enzyme/substrate interaction through PELE (Protein Energy Landscape Exploration) software (Docking).
Molecules
ΔE (Kcal/mol)
2,5 DABSA
-99.5
2,4 DABSA
-112.8
The lack of activity of POXA1b towards 2,4 DABSA seems to be due to a non correct orientation of the substrate in the active site and to the higher ΔE of 2,4 DABSA with respect to 2,5 DABSA

28. CASE STUDY
Through PELE analyses it is possible to study only the interaction of active site with the substrate. Considering the complexity of tuning laccase redox potential, the increment of the enzyme-substrate complex stability was chosen.

29. Two mutant were designed Binding Energy (Kcal/mol)
CASE STUDY
Twenty residues probably involved in 2,4 DABSA oxidation were selected through PELE simulation. All potential substitutions of these residues were analysed considering four main criteria:
Binding Energy decrease
SASA % (Solvent Accessible Surface Area) decrease
Distance between T1 copper and the substrate decrease
Quality of interaction
Two mutant were designed
Protein variant
Binding Energy (Kcal/mol)
SASA %
S:Cu1 (Å)
POXA1b wild type
-7.9
0.18
7.5
V162H_F331Y_A336N
-35.2
0.06
6.8
V162S_F331Y_A336N
-31.2
0.14
7.7

30. RESULTS OF DOCKING VS 2,4 DABSA
CASE STUDY
RESULTS OF DOCKING VS 2,4 DABSA
Wild type
V162S_F331Y_A336N
V162H_F331Y_A336N

31. RESULTS OF DOCKING VS RESORCINOL
CASE STUDY
RESULTS OF DOCKING VS RESORCINOL
Wild type
V162S_F331Y_A336N
V162H_F331Y_A336N

32. CASE STUDY
The designed mutants display an increment of affinity towards 2,4 DABSA without modifying their affinity towards Resorcinol
The designed mutants were expressed and tested on real precursors in order to validate the computational analyses.

33. Test on target precursors
CASE STUDY
Test on target precursors
Laccase
2,4 DABSA
2,5 DABSA
1,3 RESORCINOL
Mut Serine -
2.95±0.29
0.093±0.006
Mut Histidine
2.90±0.40
0.089±0.006
POXA1b
2.89±0.64
0.082±0.004
No samples have showed an increment of activity toward 2,4 DABSA respect to wild type.
If we consider that computational simulations were reliable, as confirmed by enzymes behaviour against resorcinol and 2,5 DABSA, enzyme redox potential seems to be bottleneck to overcome for 2,4 DABSA oxidation.

34. CASE STUDY
Laboratory evolution of laccases 34

35. The role of the C-terminus in basidiomycetous and ascomycetous laccases has been evaluated using either site directed and random mutagenesis.
A C-terminal protruding tail (13–14 amino acids long) has been found in the deduced amino acidic sequences of laccases from the ascomycetes. This tail is generally cleaved off by proteolysis at a conserved cleavage site to produce the active form of the enzyme.
Analysis of the 3D structure of the laccase from the fungus Melanocarpus albomyces has shown this C-terminal extension as a plug obstructing the solvent channel, thus leading to the hypothesis that its cleavage is required to favour the entrance of oxygen and the subsequent exit of water molecules.
Hakulinen N et al., (2002) Nat Struct Biol 9:601–605

36. Deletion of the last four amino acids (delDSGL559)
More recently, results obtained with Melanocarpus albomyces laccase clearly confirmed the critical role of the last amino acids in its C-terminus. The four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. C-terminal carboxylate group forms a hydrogen bond with a side chain of His coordinating to the type 3 copper.
The C-terminus penetrates to the tunnel leading towards the trinuclear site. Coppers are represented as orange balls, water atoms as red balls, and dioxygen as a red stick.
In order to analyze the role of the processed C-terminus, site-directed mutants were expressed in Trichoderma reesei and Saccharomyces cerevisiae:
Deletion of the last four amino acids (delDSGL559)
Substitutions of the last amino acid of mature protein (L559A)
Andberg M et al., (2009) FEBS J; 276:

37. Superimposition of the native enzyme (green) and the Sc(L559A) mutant structure (in blue).
Deletion of the last four amino acids dramatically affect enzyme activity, while Leu substitution reduce the turnover of the mutant proteins.
Moreover, the crystal structure of the mutant showed that the mutation of C-terminal clearly affected the trinuclear site geometry.
As a fact, owing to the lower steric limitations of the side chain of Ala if compared to that of Leu, water may occupy the space. The side chain of His140, which is coordinated to the T3 copper, rotate slightly and forme a hydrogen bond with the new water rather than with the carboxylate group of the C-terminus
Andberg M et al., (2009) FEBS J; 276:

38. Further insights in the significance of the laccase C-terminal tail have serendipitously been provided through random mutagenesis. Earlier directed evolution work on the laccase from the ascomycete Myceliophthora thermophila showed widely variable activity that was attributed to truncations of and/or mutations around its C terminus.
The open triangles mark the positions of the mutations in the most active laccase (T2). The arrows mark the protease cleavage sites relative to the processing sites of the MtL protein. The  indicates the cleavage position.
The most effective mutation (10-fold increase in total activity) adjusts the protein sequence to the different protease specificities of the heterologous host
Bulter Tet al., (2003) Appl Environ Microbiol 69:987–995

39. Whether a similar role of the C- terminal tail is possible among basidiomycete laccases too is not yet known.

40. Gelo-Pujic and co-workers reported that the redox potential of the laccase from the basidiomycete Trametes versicolor changes when its C terminus is truncated by 11 amino acids. An additional consequence of truncating the C-terminus is a reduction of the barrier to heterogeneous electron transfer.
C-terminal amino acids can affect the function of fungal laccases from basidiomycetes
Gelo-Pujic Met al., (1999) Appl Environ Microbiol 65:5515–5521

41. An unusual C-terminal extension of 16 amino acids has been found in the POXA1b laccase from Pleurotus ostreatus.
C-terminus
POXA1b
Site directed mutants POXA1bD4 and POXA1bD16 were produced in order to define a role for the C- terminal tail.
POXA1b C-terminal tail affects both catalytic performance and stability properties of the enzyme. The truncated mutants lose POXA1b peculiar stability at pH 10.
Autore F, et al., (2009) Enzyme Microb Technol 45:507–513

42. The POXA1b C-terminal tail was found mutated in one of the selected random mutants from directed evolution experiments. The P494T mutation is located in a variable and mobile loop at the C terminus.
Substitutions: L112F, P494T
C-terminal loop
Molecular dynamics simulations combined with modelling on a hybrid synthetic crystal structure from Trametes versicolor and Melanocarpus albomyces laccases were used to rationalise the functional roles of the principal mutations
Festa G., et al., (2008). Proteins; 72:25-34

43. POXA1b: three water molecules are trapped close the T2/T3 channel.
High flexibility of the C termini and involvement of these regions to direct water molecules toward the T2/T3 channel has been observed.
3M7C: three water molecules remain locked in the channel. P494T affects the position of F112, thus this is not obstructing the channel anymore. More water molecules can enter in the cavity but remain coordinated for a shorter time than in POXA1b.
Cu ions (T2/T3 cluster in green and T1 in magenta) and the water molecules in close contact with the channel of T2/T3 cluster are shown in van der Waals rendering. In POXA1b, residue L112 is highlighted, in 1M9B residue F112, and in 3M7C both residues F112 and T494 are shown.
Festa G., et al., (2008). Proteins; 72:25-34

44. CASE STUDY
Laboratory evolution of laccases

45. First Generation Library Second Generation Library
CASE STUDY
Directed Evolution of Pleurotus ostreatus laccase POXA1b
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10
1M9B I
1.5x Χ
First Generation Library
1,100 mutants
Error-Prone PCR
PoPOXA1b cDNA
Screening for improved Activity against ABTS at pH 3
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10
1L2B II
2.5x Χ √
1M10B
3M7C
Second Generation Library
1,100 mutants
Error-Prone PCR
Screening for improved Activity against ABTS at pH 3
Festa G., et al., (2008). Proteins; 72:25-34

46. CASE STUDY Directed Evolution of Pleurotus ostreatus laccase POXA1b
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10
1M9B I
1.5x Χ
Gln 37
Asn 51
Phe 112
1M10B
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10
1L2B II
2.5x Χ √
1M10B
3M7C
1M10B and 3M7C mutations were combined in the rational designed mutant, R4
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10 R4
2.5x Χ √
Thr 494
Phe 112
3M7C
Miele A et al., (2010). Submitted to Molecular Biotechnology

47. Screening for improved Activity against ABTS at pH 3
CASE STUDY
Directed Evolution of Pleurotus ostreatus laccase POXA1b
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10
1M9B I
1.5x Χ
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10
1L2B II
2.5x Χ √
1M10B
3M7C
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10 R4
2.5x Χ √
R4 Library
1,100 mutants
Error-Prone PCR
Screening for improved Activity against ABTS at pH 3
Mutant
Generation
activity
pH stability
T stability
pH3
pH5
pH7
pH10
1H6C
III
4.5x Χ √
4M10G
Miele A et al., (2010). Submitted to Molecular Biotechnology

48. The way ahead… CASE STUDY Laboratory evolution of laccases
Although some laccases are being employed successfully in industry, no natural laccase combines all the desired attributes
The way ahead…
High reduction potential
Activity towards new substrates
Stability under harsh operating conditions (-e.g. presence of organic co-solvents, extreme pH values-, thermo-stability, and others)
Stereo-selectivity