Directed Evolution of Enzymes

Overview of Directed Evolution

  • Definition: A method that requires no prior knowledge of protein structure or catalytic mechanism.

  • Purpose: To improve the 'fitness' of naturally occurring or rationally designed enzymes.

  • Mechanism: Mimics natural evolution.

  • Attributes Improved:

    • Stability

    • Specificity

    • Catalytic efficiency

Random Mutagenesis Procedure

  • Involves the following key steps:

    1. Random mutagenesis via error-prone PCR.

    2. Creation of a gene library for the protein of interest.

    3. Generation of a library of mutant genes ranging from 10210^2 to 10910^9.

    4. Insertion of DNA into a vector for protein expression.

    5. Formation of library of mutant proteins, either in cells or on the surface of viruses.

    6. Screening/selecting for a new desired property.

    7. Choosing the most 'improved' protein for further rounds of mutation.

    8. Repetition of the process using the improved variant as the new starting sequence.

Specifics of Mutagenesis

  • Error-prone PCR is often performed using Taq polymerase with an increased concentration of MgCl2 ions and the addition of MnCl2.

  • The final step in the procedure is the extraction of plasmid DNA.

Quality of Screening and Selection

  • Importance: The success of the directed evolution depends heavily on the quality of the screen or selection mechanism:

    • Screening:

    • Involves characterizing each mutant individually.

    • Selection:

    • Employs conditions that test all mutants simultaneously and permit only desirable mutants to manifest.

  • High throughput is crucial — typically 10210^2 to 10910^9 mutants need to be tested.

  • Challenges:

    • Screening for substrate binding is easy but may not be the best approach. Screening for catalysis yields better outcomes.

    • Requires methods linking enzyme activity physically to its DNA.

Screening Methods

  • In Vivo Screens: Involves changes in the phenotype of transformed microorganisms due to expression of the modified protein.

  • In Vitro Screens: Utilizing methods such as phage display.

Example of In Vivo Screening

  • Case Study: dsRed from Bevis & Glick (2002).

  • Description:

    • dsRed is a red fluorescent protein useful for fluorescence microscopy.

    • Slow maturation rate (half-time of 1111 hours) necessitated the development of a mutant with faster maturation while retaining fluorescent properties.

  • Process:

    • Generate 100,000100,000 mutants per round.

    • Measure fluorescence after 24 hours growth under light at 520extnm520 ext{nm}.

    • Select the reddest colonies for DNA isolation and repeated cycle, typically conducted 66 times.

Analysis of Mutants: Changes in DsRed.T4 Compared to Wild Type (WT)

  • Mutant Variations:

    • Mutations include: R2A, K5E, N6D, T21S, H41T, N42Q, V44A, A145P, T217A.

  • Maturation Results for the variants:

    • Relative Brightness:

    • DsRed.WT: (1.00)(1.00)

    • DsRed.T3: (0.83)(0.83)

    • DsRed.T4: (0.38)(0.38)

    • Maturation Half-time (in hours):

    • WT: 1111 hours

    • T3: 1.31.3 hours

    • T4: 0.710.71 hours

    • Quantum Yield:

    • WT: 0.680.68

    • T3: 0.590.59

    • T4: 0.440.44

In Vitro Screening via Phage Display

  • Procedure:

    1. A library of DNA fragments cloned with the gene encoding g3p is transformed into E.coli.

    2. Helper phage infection is necessary to produce phage particles.

    3. Panning Process: Carried out 3−53-5 times with increasing stringency in washing procedures.

    4. Creation of a library of DNA variants demonstrating high binding affinities.

  • The phagemids are retransformed into E.coli for DNA sequencing or to generate new libraries of mutants via error-prone PCR.

High-Throughput Screening and Smart Libraries

  • Very large libraries (up to 10910^9 mutants) have been demonstrated to enhance enzyme fitness.

  • Enzyme stability tends to improve significantly, while substrate specificity is more challenging but can be achieved (example: Cytochrome P450 modifications).

  • Smart Libraries:

    • In situations where high-throughput is unattainable, smaller libraries focused on specific targeted residues are generated, called smart libraries.

    • Hot-Spots: Identified sites where mutations should be concentrated based on prior knowledge, structural data, or computational modeling.

    • Saturation Mutagenesis can be implemented alongside random mutagenesis cycles.

Successful Case Study in Catalyst Development

  • Directed evolution of the Kemp elimination catalyst leads to a >200 fold increase in catalytic efficiency (discriminated by k<em>cat/K</em>mk<em>{cat}/K</em>m):

    • Example of Mutants:

    • Mutant KE07: k<em>cat=0.018exts−1,Km=1.4extmM,k</em>cat/Km=12.2extM−1exts−1k<em>{cat} = 0.018 ext{s}^{-1}, Km = 1.4 ext{mM}, k</em>{cat}/K_m = 12.2 ext{M}^{-1} ext{s}^{-1}

    • R7 Mutant: k<em>cat=1.37exts−1,Km=0.54extmM,k</em>cat/Km=2590extM−1exts−1k<em>{cat} = 1.37 ext{s}^{-1}, Km = 0.54 ext{mM}, k</em>{cat}/K_m = 2590 ext{M}^{-1} ext{s}^{-1}

Development of Catalytic Antibodies (Abzymes)

  • Definition: Enzymes created with antibodies that recognize and stabilize transition states (TS).

  • Key Principle:

    • Enzymes stabilize the transition state more effectively than they do the substrate.

    • In 1946, Linus Pauling highlighted that to catalyze a reaction, an enzyme must stabilize the TS selectively.

Catalytic Antibody Production Protocol

  • Process:

    1. Synthesize Transition State Analogue (TSA).

    2. Covalently link the TSA to a carrier protein.

    3. Immunize a mouse with the complex.

    4. Isolate B-cells from the mouse spleen and fuse with myeloma cells to yield immortal antibody-expressing hybridoma cells.

    5. Screen monoclonal antibodies for catalytic activity.

Transition State Analogues (TSA)

  • TSs are inherently unstable and cannot be isolated.

  • TSA: a stable structure approximating the TS is used for antibody synthesis.

  • The success of the interaction depends on how accurately the TSA mimics the TS.

Examples of Transition State Analogues

  1. Ester Hydrolysis:

    • TSA represents unstable TS of ester hydrolysis reactions.

  2. Metal Insertion into the Porphyrin Ring:

    • A TSA mimicking the transition state for metal ion insertion into the porphyrin ring can raise catalytic efficiencies comparable to natural enzymes.

Applications of Catalytic Antibodies

  1. Novel Catalysis:

    • Catalytic antibodies for aldol reactions facilitate carbon-carbon bond formation.

  2. Drug-Clearance (Cocaine Hydrolysis):

    • Humanized antibodies breaking down cocaine for overdose treatment.

    • Benefits include recycling of the catalytic antibodies within the bloodstream, enhancing detoxification capabilities.

  3. Pro-drug Activation:

    • Use of catalytic antibodies for antibody-directed enzyme pro-drug therapy.

Performance of Catalytic Antibodies

  • Over 100100 catalytic antibodies described with following performance metrics:

    • Catalytic Efficiency: 106−108extM−1exts−110^6 - 10^8 ext{M}^{-1} ext{s}^{-1}

    • Rate Enhancement 106−101210^6 - 10^{12} compared to uncatalyzed reactions.

    • Affinity for TS: 10−8−10−23extM−110^{-8} - 10^{-23} ext{M}^{-1}

Limitations and Strategies for Improvement of Catalytic Antibodies

  • Limitations include:

    • Imperfect TSA design lacking catalytic machinery.

    • Rigid structure dynamics of antibodies compared to enzymes.

  • Strategies for improvement:

    • Utilize site-directed mutagenesis to introduce catalytic residues into the antibodies' binding sites.

    • Employ directed evolution to boost catalytic efficiency further.