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PART Ⅰ ENZYME FUNCTION3

1 A Short Practical Guide to the Quantitative Analysis of Engineered Enzymes&Christopher D.Bayer and Florian Hollfelder3

1.1 Introduction3

1.2 Quantifying Reaction Progress4

1.3 Typical Saturation Plots Give Michaelis-Menten Parameters5

1.4 What Can Go Wrong？8

1.5 Dealing with Multiphasic and Pre-Steady-State Kinetics12

1.6 Evaluating Enzymes16

2 Protein Conformational Motions：Enzyme Catalysis&Xinyi Huang，C.Tony Liu，and Stephen J.Benkovic21

2.1 Introduction21

2.2 Multidimensional Protein Landscape and the Timescales of Motions22

2.3 Conformational Changes in Enzyme-Substrate Interactions26

2.4 Conformational Changes in Catalysis28

2.4.1 Protein Dynamics of DHFR in the Catalytic Cycle30

2.4.2 Temporally Overlap：Correlation Does Not Mean Causation32

2.4.3 Fast Timescale Conformational Fluctuations34

2.4.4 Effect of Conformational Changes on the Electrostatic Environment36

2.5 Conservation of Protein Motions in Evolution38

2.6 Designing Protein Dynamics39

2.7 Concluding Remarks40

3 Enzymology Meets Nanotechnology：Single-Molecule Methods for Observing Enzyme Kinetics in Real Time&Kerstin G.Blank，Anna A.Wasiel，and Alan E.Rowan47

3.1 Introduction48

3.2 Single-Turnover Detection53

3.2.1 Fluorescent Reporter Systems53

3.2.2 Measurement Setup56

3.2.3 Data Analysis57

3.3 Single-Enzyme Kinetics60

3.3.1 Candida antarctica Lipase B63

3.3.2 Thermomyces lanuginosus Lipase67

3.3.3 α-Chymotrypsin73

3.3.4 Nitrite Reductase78

3.3.5 Summary84

3.4 New Developments Facilitated by Nanotechnology88

3.4.1 Nano-optical Approaches89

3.4.2 Nano-electronic Approaches96

3.4.3 Nanomechanical Approaches103

3.4.4 Summary108

3.5 Conclusion110

4 Interfacial Enzyme Function Visualized Using Neutron，X-Ray，and Light-Scattering Methods&Hanna Wacklin and Tommy Nylander125

4.1 Phospholipase A2：An Interfacially Activated Enzyme126

4.1.1 Neutron Reflection129

4.1.2 Ellipsometry130

4.1.3 Activity of Naja mossambica mossambica PLA2130

4.1.4 Fate of the Reaction Products133

4.1.5 The Lag Phase and Activation of Pancreatic PLA2135

4.1.6 Distribution of Products during the Lag Phase138

4.1.7 Hydrolysis of DPPC by Pancreatic PLA2139

4.1.8 Role of the Reaction Products in PLA2 Activation141

4.1.9 Effect of pH and Activation by Me-β-cyclodextrin144

4.2 Other Lipolytic Enzyme Reactions on Surfaces150

4.2.1 Triacylglycerol Lipases and the Role of Lipid Liquid Crystalline Nanostructures150

4.3 Cellulase Enzymes154

4.4 Conclusion158

5 Folding Dynamics and Structural Basis of the Enzyme Mechanism of Ubiquitin C-Terminal Hydroylases&Shang-Te Danny Hsu167

5.1 Introduction169

5.1.1 UCH-L1171

5.1.1.1 Genetic association between UCH-L1 and neurodegenerative diseases171

5.1.1.2 UCH-L1 in oncogenesis175

5.1.2 Molecular Insights into the Pathogenesis Associated with UCH-L1175

5.1.3 UCHL3177

5.1.4 UCHL5178

5.1.5 BAP1179

5.2 UCH Structures180

5.3 Folding Dynamics and Kinetics183

5.4 Substrate Recognition184

5.5 Enzyme Mechanism186

5.6 Conclusion189

6 Stabilization of Enzymes by Metal Binding：Structures of Two Alkalophilic Bacillus Subtilases and Analysis of the Second Metal-Binding Site of the Subtilase Family&Jan Dohnalek，Katherine E.McAuley，Andrzej M.Brzozowski，Peter R.φstergaard，Allan Svendsen，and Keith S.Wilson203

6.1 Introduction：Subtilases and Metal Binding203

6.1.1 Calcium-Binding Sites in Bacillus：Proposal for a Standard Nomenclature209

6.1.2 The Weak Metal-Binding Site214

6.2 Two New Structures of Subtilases with Altered Calcium Sites216

6.2.1 Proteinase SubTY216

6.2.1.1 The overall fold216

6.2.1.2 The active site216

6.2.1.3 SubTY calcium and sodium sites218

6.2.1.4 SubTY disulfide bridge219

6.2.2 SubHal220

6.2.2.1 The unliganded form of SubHal220

6.2.2.2 The SubHal：CI2A complex221

6.2.2.3 Termini，surface，and pH stability of SubHal221

6.2.2.4 The two crystallographically independent SubHal：CI2A complexes223

6.2.2.5 The calcium sites in SubHal224

6.2.2.6 The active site of SubHal226

6.2.3 Enzymatic Activity of SubTY and SubHal228

6.2.4 Comparison of SubTY and SubHal with Other Subtilases228

6.2.5 The SubHal C-domain Compared to the Eukaryotic PCs，Furin and Kexin232

6.2.5.1 Active site comparison233

6.2.5.2 The specificity pockets234

6.2.5.3 Inhibitor CI2A binding234

6.2.6 Activity Profiles236

6.2.7 Comparison of Metal Binding at the Strong and Weak Sites in the S8 Family236

6.2.8 The Ca-Ⅱ and Na-Ⅱ Metal-Binding Sites237

6.3 Conclusion：Implications for Structural Studies of Enzymes248

6.4 Materials and Methods249

6.4.1 SubTY249

6.4.1.1 Protein production and purification249

6.4.1.2 Purification of the SubTY：CI2A （1：1）complex250

6.4.1.3 Crystallization250

6.4.1.4 Structure determination 2516.4.2 SubHal251

6.4.2.1 Protein production and purification251

6.4.2.2 Purification of the SubHal：CI2A （1：1）complex252

6.4.2.3 Crystallization252

6.4.2.4 Structure determination253

6.4.3 Protease Assays256

6.4.4 pH Stability257

6.4.5 Data Deposition257

7 Structure and Functional Roles of Surface Binding Sites in Amylolytic Enzymes&Darrell Cockburn and Birte Svensson267

7.1 Introduction267

7.2 Identifiication of SBSs：X-Ray Crystallography271

7.3 Bioinformatics of SBS Enzymes273

7.4 Binding Site Isolation275

7.5 Protection of Binding Sites from Chemical Labeling277

7.6 Nuclear Magnetic Resonance277

7.7 Binding Assays278

7.8 Activity Assays282

7.9 Future Prospects283

7.10 Conclusion286

8 Interfacial Enzymes and Their Interactions with Surfaces：Molecular Simulation Studies&Nathalie Willems，Mickael Lelimousin，Heidi Koldsφ，and Mark S.P.Sansom297

8.1 Introduction297

8.2 Enzyme Interactions at Interfaces299

8.3 Molecular Dynamic Simulations of Biomolecular Systems301

8.4 Lipases303

8.4.1 Atomistic MD Studies of Lipase Interactions with Interfaces304

8.4.2 The Role of Water in Lipase Catalysis at Interfaces307

8.5 Coarse-Grained MD Studies of Interfacial Enzymes：Orientation and Interactions309

8.5.1 Phospholipase A2309

8.5.2 PTEN310

8.6 Conclusions311

PART Ⅱ ENZYME DESIGN321

9 Sequence，Structure，Function：What We Learn from Analyzing Protein Families&Michael Widmann and Jurgen Pleiss321

9.1 Introduction321

9.2 Detection of Inconsistencies Utilizing a Standard Numbering Scheme323

9.3 Identification of Functionally Relevant Positions327

9.4 The Modular Structure of Thiamine Diphosphate-Dependent Decarboxylases330

9.5 Stereoselectivity-Determining Positions：The S-Pocket Concept in Thiamine Diphosphate-Dependent Decarboxylases333

9.6 Regioselectivity-Determining Positions：Design of Smart Cytochrome P450 Monooxygenase Libraries336

9.7 Substrate Specificity-Determining Positions：The GX/GGGX Motif in Lipases340

9.8 Conclusion341

10 Bioinformatic Analysis of Protein Families to Select Function-Related Variable Positions&Dmitry Suplatov，Evgeny Kirilin，and Vytas Svedas351

10.1 Introduction352

10.2 Bioinformatic Analysis of Evolutionary Information to Identify Function-Related Variable Positions359

10.2.1 Problem Definition359

10.2.2 Scoring Schemes in the Variable Position Selection：High-Entropy，Subfamily-Specific，and Co-Evolving Positions361

10.2.3 Association of the Variable Positions with Functional Subfamilies366

10.2.4 How to Select Functionally Important Positions as Hotspots for Further Evaluation：Implementation of Statistical Analysis366

10.3 The Bioinformatic Analysis of Diverse Protein Superfamilies369

10.3.1 Bioinformatic Challenges at Studying Enzymes369

10.3.2 Zebra：A New Algorithm to Select Functionally Important Subfamily-Specific Positions from Sequence and Structural Data370

10.4 Subfamily-Specific Positions as a Tool for Enzyme Engineering375

10.5 Conclusion377

11 Decoding Life Secrets in Sequences by Chemicals&Zizhang Zhang387

11.1 Introduction388

11.2 Linking an Enzyme’s Activity to Its Sequence389

11.3 Refiining the Sequence Space to a Specifiic Function by Directed Evolution395

11.4 Linking Chemistry to -Omics with High-Throughput Screening Methods398

11.5 Finding Large Sequence Space of a Specific Function from Microbial Diversity400

11.6 Linking Sequences to Substromes at the Molecular Level404

11.6.1 Biocatalytic Study of EHs405

11.6.2 Pharmacological Study of EHs407

11.6.3 Mechanistic Study of EHs407

11.6.4 What We Have Learned from the Studies of EH410

11.6.5 Technologies with Potentials in Genochemistry Approach410

11.7 Correlating with Computational Methods410

11.8 Problems That Genochemistry Can Potentially Tackle413

11.9 Conclusion 41412 Role of Tunnels and Gates in Enzymatic Catalysis&Sergio M.Marques，Jan Brezovsky，and Jiri Damborsky421

12.1 Introduction421

12.2 Protein Tunnels423

12.2.1 Structural Basis and Function423

12.2.2 Identification Methods427

12.2.3 Molecular Engineering429

12.3 Protein Gates431

12.3.1 Structural Basis and Function431

12.3.2 Identification Methods437

12.3.3 Molecular Engineering440

12.4 Conclusions442

13 Molecular Descriptors for the Structural Analysis of Enzyme Active Sites&Valerio Ferrario，Lydia Siragusa，Cynthia Ebert，Gabriele Cruciani，and Lucia Gardossia465

13.1 Introduction：Molecular Descriptors for Investigation of Enzyme Catalysis465

13.2 Molecular Descriptors Based on Molecular Interaction Fields467

13.3 Multivariate Statistical Analysis for Processing and Interpretation of Molecular Descriptors472

13.4 Grind Descriptors for the Study of Substrate Specificity475

13.5 VolSurf Descriptors for the Modeling of Substrate Specifiicity477

13.6 Differential MIFs Descriptors for the Study of Enantioselectivity479

13.7 Hybrid MIFs Descriptors for the Computation of Entropic Contribution to Enantiodiscrimination481

13.8 Analysis of Enzyme Active Sites for Rational Enzyme Engineering484

13.9 BioGPS Descriptors for in silico Rational Design and Screening of Enzymes489

13.10 Conclusions495

14 Hydration Effects on Enzyme Properties in Nonaqueous Media Analyzed by MD Simulations&Diana Lousa，Antonio M.Baptista，and Claudio M.Soares501

14.1 Enzyme Reactions in Nonaqueous Solvents502

14.2 Classes of Nonaqueous Solvents503

14.3 The Role of Water in Nonaqueous Biocatalysis504

14.4 Effect of Water Content on Enzyme Structure and Dynamics504

14.5 Effect of Water Content on Enzyme Selectivity507

14.6 Hydration Mechanisms of Enzymes in Polar and Nonpolar Solvents508

14.7 Enzyme Behavior as a Function of Water Activity510

14.8 Hydration Effects on Enzyme Reactions in Ionic Liquids512

14.9 Hydration Effects on Enzyme Reactions in Supercritical Fluids514

14.10 Conclusions516

15 Understanding Esterase and Amidase Reaction Specificities by Molecular Modeling&Per-Olof Syren523

15.1 Introduction523

15.2 Fundamental Catalytic Concepts525

15.2.1 Fundamental Chemistry of Amides and Esters525

15.2.2 Esterases and Amidases and Their Metabolic Significance525

15.2.3 Fundamental Chemical Aspects of Amidase and Esterase Catalysis526

15.2.4 Impact of Stereoelectronic Effects on the Enzymatic Reaction Mechanism529

15.3 Molecular Modeling of Fundamental Catalytic Concepts529

15.3.1 QM Calculations on Amidases and Esterases529

15.3.2 MD Simulations on Amidases and Esterases535

15.3.3 QM/MM Simulations on Amidases and Esterases539

15.4 Outlook and Implications for Enzyme Design544

15.5 Additional Comments546

PART Ⅲ ENZYME DIVERSITY561

16 Toward New Nonnatural TIM-Barrel Enzymes Using Computational Design and Directed Evolution Approaches&Mirja Krause and Rik K.Wierenga561

16.1 Introduction562

16.2 General Aspects of Protein Engineering566

16.2.1 Library Creation Methods569

16.2.2 Structure-Based Library Design572

16.2.3 Optimal Libraries for Directed Evolution Methods574

16.2.4 Data-Driven Design （Semirational Design）578

16.2.5 Protein Engineering by Selection and Screening Methods579

16.3 Directed Evolution Studies with TIM-Barrel Enzymes584

16.3.1 Protein Engineering Studies of TIM-Barrel Proteins586

16.3.2 The Kemp Eliminases590

16.4 Concluding Remarks596

17 Handling the Numbers Problem in Directed Evolution&Carlos G.Acevedo-Rocha and Manfred T.Reetz613

17.1 Introduction614

17.2 Saturation Mutagenesis in Directed Evolution617

17.3 Statistical Analyses620

17.3.1 Conventional Statistics Based on the Patrick and Firth Algorithm620

17.3.2 Statistics Based on the Nov Algorithm624

17.4 How to Group and Randomize Amino Acid Positions626

17.5 Fitness Landscapes628

17.5.1 Fujiyama vs.Badlands Fitness Landscapes628

17.5.2 Fitness-Pathway Landscapes and How to Escape from Local Minima630

17.6 Conclusions and Perspectives636

18 Hints from Nature：Metagenomics in Enzyme Engineering&Esther Gabor，Birgit Heinze，and Jurgen Eck643

18.1 Metagenomics and the Ideal Enzyme644

18.2 Molecular Microdiversity647

18.3 Metagenomic Enzyme Chimera650

18.4 Outlook653

19 A Functional and Structural Assessment of Circularly Permuted Bacillus circulans Xylanase and Candida antarctica Lipase B&Stephan Reitinger and Ying Yu657

19.1 Introduction657

19.2 Naturally Occurring Circular Permutations：Selected Examples658

19.3 Circular Permutation of Bacillus circulans Xylanase661

19.4 Circular Permutation on Candida antarctica Lipase B669

19.5 Conclusion674

20 Ancestral Reconstruction of Enzymes&Satoshi Akanuma and Akihiko Yamagishi683

20.1 Introduction683

20.2 Reconstruction of an Ancestral Protein Sequence684

20.2.1 Overview684

20.2.2 Methods for Ancestral Sequence Reconstruction684

20.2.3 Early Works686

20.3 The Commonote687

20.3.1 The Last Universal Common Ancestor，the Commonote687

20.3.2 Theoretical Studies on the Environmental Temperature of the Commonote688

20.3.3 Reconstruction of an Ancestral Nucleoside Diphosphate Kinase689

20.3.4 Estimation of the Environmental Temperature of the Commonote692

20.4 Application to Designing Thermally Stable Proteins693

20.4.1 Design of Thermally Stable Proteins693

20.4.2 Case Studies to Create Thermally Stable Enzymes by Introducing Ancestral Residues as Amino Acid Substitutions694

20.4.3 Reconstruction of Thermally Stable，Ancestral DNA Gyrase Using a Small Set of Homologous Amino Acid Sequences696

20.5 Conclusion697

PART Ⅳ ENZYME SCREENING AND ANALYSIS707

21 High-Throughput Screening or Selection Methods for Evolutionary Enzyme Engineering&Shuobo Shi，Hongfang Zhang，Ee Lui Ang，and Huimin Zhao707

21.1 Introduction708

21.2 Selection710

21.2.1 Solid-Medium-Based Selection717

21.2.2 Liquid-Medium-Based Selection719

21.2.3 Display-Based Selection722

21.3 Screening724

21.3.1 Chromatography- and Mass-Spectrometry-Based Screening725

21.3.2 Solid-Medium-Based Screening726

21.3.3 Microtiter-Plate-Based Screening727

21.3.4 Yeast Two-/Three-Hybrid System729

21.3.5 FACS-Based Screening729

21.3.6 Microfluidics-Based Screening732

21.4 Conclusions and Prospects734

22 Nanoscale Enzyme Screening Technologies&Helen Webb-Thomasen and Andreas H.Kunding745

22.1 Introduction745

22.2 Approaches to Nanocompartmentalization of Enzymes746

22.2.1 Liposomes747

22.2.1.1 Addressability747

22.2.1.2 Reagent exchange749

22.2.2 Polymersomes and VirusLike Particles751

22.2.3 Water-in-Oil Emulsion Droplets752

22.2.3.1 Addressability755

22.2.3.2 Reagent exchange755

22.3 Microfabricated Chip Devices for Enzyme Compartmentalization and Screening756

22.3.1 Microfluidic-Generated Emulsion Droplets757

22.3.2 Microfabricated Arrays762

22.3.2.1 Optical fiber microarrays762

22.3.2.2 Elastomeric microarrays763

22.3.2.3 Surface tension microarrays765

22.4 Conclusion and Current Challenges767

22.5 Future Improvements769

23 Computational Enzyme Engineering：Activity Screening Using Quantum Chemistry&Martin R.Hediger777

23.1 Motivation778

23.2 Introduction779

23.3 Methods780

23.3.1 Calculation Engines780

23.3.2 Molecular Modeling782

23.3.3 Software786

23.4 Applications786

23.4.1 Overview786

23.4.2 Engineering Candida antarctica Lipase B787

23.4.3 Engineering Bacillus circulans Xylanase793

23.5 Conclusions800

24 In Silico Screening of Enzyme Variants by Molecular Dynamics Simulation&Hein J.Wijma805

24.1 Potential Applications of MD Simulations For Improving Enzymes805

24.2 Molecular Dynamics vs.Other in silico Methods809

24.3 Improving Catalytic Activity by MD Screening812

24.3.1 Transition-State Simulation812

24.3.2 High-Energy Intermediate Simulation814

24.3.3 Substrate Simulation with Near-Attack Conformations815

24.3.4 Substrate Simulation with Monitoring of H Bonds817

24.4 Predicting and Improving Binding Affiinity818

24.5 MD Screening to Improve Enzyme Stability819

24.6 Improving Correlation between MD and Experiment822

24.6.1 Force Field Inaccuracies822

24.6.2 Sampling Concerns823

24.6.3 Other Concerns824

24.7 Outlook and Further Possibilities825

25 Kinetic Stability of Variant Enzymes&Jose M.Sanchez-Ruiz835

25.1 Kinetics vs&Thermodynamics in Protein Stability835

25.2 Mutation Effects on Kinetic Stability：A Description Based on the Transition State for Irreversible Denaturation838

25.3 Kinetic Stability Linked to the Breakup of Interactions in the Transition State：Pro-dependent Proteases841

25.4 Kinetic Stability Linked to Substantially Unfolded Transition States：Thioredoxin and Phytase Enzymes842

25.5 Role of Solvation Barriers in Kinetic Stability：Lipases and Triose Phosphate Isomerases848

25.6 Concluding Remarks852

Index859