Directed molecular evolution of proteins : or how to improve enzymes for biocatalysis / edited by Susanne Brakmann and Kai Johnsson.

Format:

Book

Contributor:

Brakmann, Susanne.

Johnsson, Kai.

Language:

English

Subjects (All):

Proteins--Evolution.

Proteins.

Proteins--Chemical modification.

Molecular evolution.

Evolution, Molecular.

Medical Subjects:

Proteins.

Evolution, Molecular.

Physical Description:

xi, 357 pages : illustrations (some color) ; 25 cm

Place of Publication:

Weinheim : Wiley-VCH, 2002.

Summary:

The book presented here focuses on the directed evolution of proteins, which has established itself as a powerful method for designing enzymes showing new substrate specificities. It includes a comprehensive repertoire of techniques for producing combinatorial enzyme libraries, while the functional gene expression in a suitable host helps in selecting the appropriate structure, making fast screening a necessity. This book illustrates both the theoretical background as well as the potential of this interesting method in practice - which is becoming ever more important even in classical organic synthesis!

Contents:

2 Evolutionary Biotechnology

From Ideas and Concepts to Experiments and Computer Simulations 5

2.1 Evolution in vivo

From Natural Selection to Population Genetics 5

2.2 Evolution in vitro

From Kinetic Equations to Magic Molecules 8

2.3 Evolution in silico

From Neutral Networks to Multi-stable Molecules 16

2.4 Sequence Structure Mappings of Proteins 25

3 Using Evolutionary Strategies to Investigate the Structure and Function of Chorismate Mutases 29

3.2 Selection versus Screening 30

3.2.1 Classical solutions to the sorting problem 31

3.2.2 Advantages and limitations of selection 32

3.3 Genetic Selection of Novel Chorismate Mutases 33

3.3.1 The selection system 35

3.3.2 Mechanistic studies 37

3.3.2.1 Active site residues 37

3.3.2.2 Random protein truncation 42

3.3.3 Structural studies 44

3.3.3.1 Constraints on interhelical loops 44

3.3.4 Altering protein topology 46

3.3.4.1 New quaternary structures 47

3.3.4.2 Stable monomeric mutases 49

3.3.5 Augmenting weak enzyme activity 51

3.3.6 Protein design 53

3.4 Summary and General Perspectives 57

4 Construction of Environmental Libraries for Functional Screening of Enzyme Activity 63

4.1 Sample Collection and DNA Isolation from Environmental Samples 65

4.2 Construction of Environmental Libraries 68

4.3 Screening of Environmental Libraries 71

5 Investigation of Phage Display for the Directed Evolution of Enzymes 79

5.2 The Phage Display 79

5.3 Phage Display of Enzymes 81

5.3.1 The expression vectors 81

5.3.1.1 Filamentous bacteriophages 81

5.3.1.2 Other phages 83

5.3.2 Phage-enzymes 84

5.4 Creating Libraries of Mutants 87

5.5 Selection of Phage-enzymes 89

5.5.1 Selection for binding 89

5.5.2 Selection for catalytic activity 90

5.5.2.1 Selection with substrate or product analogues 90

5.5.2.2 Selection with transition-state analogues 92

5.5.2.3 Selection of reactive active site residues by affinity labeling 96

5.5.2.4 Selection with suicide substrates 98

5.5.2.5 Selections based directly on substrate transformations 102

6 Directed Evolution of Binding Proteins by Cell Surface Display: Analysis of the Screening Process 111

6.2 Library Construction 113

6.2.1 Mutagenesis 113

6.2.2 Expression 114

6.3 Mutant Isolation 115

6.3.1 Differential labeling 115

6.3.2 Screening 119

8 Advanced Screening Strategies for Biocatalyst Discovery 159

8.2 Semi-quantitative Screening in Agar-plate Formats 161

8.3 Solution-based Screening in Microplate Formats 164

8.4 Robotics and Automation 169

9 Engineering Protein Evolution 177

9.2 Mechanisms of Protein Evolution in Nature 178

9.2.1 Gene duplication 179

9.2.2 Tandem duplication 180

[beta alpha]-barrels 181

9.2.3 Circular permutation 182

9.2.4 Oligomerization 183

9.2.5 Gene fusion 184

9.2.6 Domain recruitment 184

9.2.7 Exon shuffling 186

9.3 Engineering Genes and Gene Fragments 187

9.3.1 Protein fragmentation 188

9.3.2 Rational swapping of secondary structure elements and domains 189

9.3.3 Combinatorial gene fragment shuffling 190

9.3.4 Modular recombination and protein folding 194

9.3.5 Rational domain assembly

engineering zinc fingers 199

9.3.6 Combinatorial domain recombination

exon shuffling 200

9.4 Gene Fusion

From Bi- to Multifunctional Enzymes 203

9.4.1 End-to-end gene fusions 203

9.4.2 Gene insertions 203

9.4.3 Modular design in multifunctional enzymes 204

9.5 Perspectives 208

10 Exploring the Diversity of Heme Enzymes through Directed Evolution 215

10.2 Heme Proteins 216

10.3 Cytochromes P450 218

10.3.1 Mechanism 220

10.3.2.1 The catalytic cycle 220

10.3.2.2 Uncoupling 222

10.3.2.3 Peroxide shunt pathway 222

10.4 Peroxidases 223

10.4.2 Mechanism 223

10.4.2.1 Compound I formation 223

10.4.2.2 Oxidative dehydrogenation 226

10.4.2.3 Oxidative halogenation 226

10.4.2.4 Peroxide disproportionation 226

10.4.2.5 Oxygen transfer 227

10.5 Comparison of P450s and Peroxidases 227

10.6 Chloroperoxidase 228

10.7 Mutagenesis Studies 229

10.7.1 P450s 230

10.7.1.1 P450[subscript cam] 230

10.7.1.2 Eukaryotic P450s 230

10.7.2 HRP 231

10.7.3 CPO 231

10.7.4 Myoglobin (Mb) 232

10.8 Directed Evolution of Heme Enzymes 233

10.8.1 P450s 233

10.8.2 Peroxidases 234

10.8.3 CPO 236

10.8.4 Catalase I 236

10.8.5 Myoglobin 237

10.8.6 Methods for recombination of P450s 237

11 Directed Evolution as a Means to Create Enantioselective Enzymes for Use in Organic Chemistry 245

11.2 Mutagenesis Methods 247

11.3 Overexpression of Genes and Secretion of Enzymes 248

11.4 High-Throughput Screening Systems for Enantioselectivity 250

11.5 Examples of Directed Evolution of Enantioselective Enzymes 257

11.5.1 Kinetic resolution of a chiral ester catalyzed by mutant lipases 257

11.5.2 Evolution of a lipase for the stereoselective hydrolysis of a meso-compound 268

11.5.3 Kinetic resolution of a chiral ester catalyzed by a mutant esterase 269

11.5.4 Improving the enantioselectivity of a transaminase 270

11.5.5 Inversion of the enantioselectivity of a hydantoinase 270

11.5.6 Evolving aldolases which accept both D- and L-glyceraldehydes 271

12 Applied Molecular Evolution of Enzymes Involved in Synthesis and Repair of DNA 281

12.2 Directed Evolution of Enzymes 282

12.2.1 Site-directed mutagenesis 283

12.2.2 Directed evolution 284

12.2.3 Genetic damage 285

12.2.4 PCR mutagenesis 286

12.2.5 DNA shuffling 287

12.2.6 Substitution by oligonucleotides containing random mutations (random mutagenesis) 288

12.3 Directed Evolution of DNA polymerases 289

12.3.1 Random mutagenesis of Thermus aquaticus DNA Pol I 291

12.3.1.1 Determination of structural components for Taq DNA polymerase fidelity 292

12.3.1.2 Directed evolution of a RNA polymerase from Taq DNA polymerase 293

12.3.1.3 Mutability of the Taq polymerase active site 294

12.3.2 Random oligonucleotide mutagenesis of Escherichia coli Pol I 294

12.4 Directed Evolution of Thymidine Kinase 295

12.5 Directed Evolution of Thymidylate Synthase 297

12.6 O[superscript 6]-Alkylguanine-DNA Alkyltransferase 300

13 Evolutionary Generation versus Rational Design of Restriction Endonucleases with Novel Specificity 309

13.1.1 Biology of restriction/modification systems 309

13.1.2 Biochemical properties of type II restriction endonucleases 310

13.1.3 Applications for type II restriction endonucleases 311

13.1.4 Setting the stage for protein engineering of type II restriction endonucleases 313

13.2 Design of Restriction Endonucleases with New Specificities 313

13.2.1 Rational design 313

13.2.1.1 Attempts to employ rational design to change the specificity of restriction enzymes 313

14.2.1.1 Changing the substrate specificity of type IIs restriction enzymes by domain fusion 316

13.2.1.3 Rational design to extend specificities of type II restriction enzymes 316

13.2.2 Evolutionary design of extended specificities 318

13.3 Summary and Outlook 324

14 Evolutionary Generation of Enzymes with Novel Substrate Specificities 329.

Notes:

Includes bibliographical references and index.

ISBN:

3527304231

OCLC:

48153128