Terpene Synthases / edited by Jeffrey Rudolf.

Format:

Book

Contributor:

Rudolf, Jeffrey, editor.

Series:

Methods in enzymology ; 699.

Methods in enzymology ; 699

Language:

English

Subjects (All):

Terpenes.

Enzymes.

Physical Description:

1 online resource (538 pages)

Edition:

First edition.

Place of Publication:

Cambridge, MA : Academic Press, [2024]

Summary:

Terpene Synthases, Volume 700 in the Methods in Enzymology series, continues the legacy of this highly respected laboratory standard with its first dedicated collection on this important family of enzymes.Terpene synthases are a diverse set of enzymes that use exquisite mechanisms to form complex (poly)cyclic hydrocarbon skeletons.

Contents:

Front Cover

Series Page

Methods in Enzymology

Copyright

Contents

Contributors

Preface

Chapter One: Methods for the preparation and analysis of a bifunctional class II diterpene synthase, copalyl diphosphate synthase from Penicillium fellutanumPreparation and analysis of synthase from Penicillium fellutanum

1 Introduction

2 Expression and purification of PfCPS

2.1 Equipment

2.2 Materials

2.3 Transformation procedure

2.4 Expression and purification procedure

3 Preparation of cryo-EM grids

3.1 Equipment

3.2 Materials

3.3 Cryo-EM grid preparation procedure

4 Data analysis

4.1 Software

4.2 Cryo-EM data processing

5 Summary

Acknowledgments

References

Chapter Two: Deciphering magnesium binding site and structure-function insights in a class II sesquiterpene cyclase

2 General method and statistical analysis

3 Genome mining for the discovery of the specific class II STC

3.2 Procedures

3.3 Notes

4 In vivo expression and product identification

4.1 Equipment

4.2 Reagents

4.3 Procedures

4.4 Notes

5 Expression of the protein and in vitro activity test

5.1 Equipment

5.2 Reagents

5.3 Procedures

5.3.1 Procedures for protein overexpression

5.3.2 Procedures for in vitro activity tests

5.4 Notes

6 Crystallization of target protein

6.1 Equipment

6.2 Reagents

6.3 Procedures

6.3.1 Crystal growth screening

6.3.2 Calibrate protein standard curve

6.3.3 Collecting protein crystals and diffraction data

6.4 Notes

7 Relative activity test and kinetics of the wild-type protein and its variants

7.1 Equipment

7.2 Reagents

7.3 Procedures

7.3.1 Construct the mutants into expression vector

7.3.2 In vitro test for relative activity and kinetic analysis

7.4 Notes.

8 Summary and conclusions

Acknowledgements

Chapter Three: Structural biology of terpene synthases

2 Gene clone and plasmid construction

2.2 Reagents

2.3 Procedure

2.4 Notes

3 Protein purification

3.2 Buffers and reagents

3.3 Procedure

3.4 Notes

4 Crystallization

4.2 Crystallization kits and reagents

4.3 Procedure

5 Data collection and structural elucidation

5.2 Procedure

5.3 Notes

6 Summary and conclusions

Chapter Four: Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase

2 Construct design

2.4 Sequences

3 Expression and purification

4 Steady-state kinetics

4.2 Materials

5 Product analysis by gas chromatography-mass spectrometry (GC-MS)

5.2 Materials

5.3 Procedure

6 Grid preparation for cryo-EM

6.2 Materials

6.3 Procedure

7 Summary

Chapter Five: Translational fusion of terpene synthases for metabolic engineering: Lessons learned and practical considerations

2.1 Choice of terpene synthase

2.2 Choice of fusion partner

2.3 Protein orientation

2.4 Linker properties

2.5 Promoter type and strength

2.6 Vector for pathway expression

2.7 Cloning strategy

3 Experimental design

3.1 Choice of base strain

3.2 Experimental controls

3.3 Fermentation conditions

3.4 Number of replicates

3.5 Sampling times

4 Detailed workflow

4.1 Materials and equipment checklist.

4.1.1 Materials and equipment for vector cloning

4.1.2 Materials and equipment for yeast transformation

4.1.3 Materials and equipment for fermentation

4.1.4 Materials and equipment for metabolomics

4.1.5 Materials and equipment for proteomics

4.1.6 Recipes of common media and buffers

4.2 Vector cloning

4.3 Transformation

4.4 Fermentation

4.5 Metabolomics

4.6 Proteomics

5 Conclusions

Chapter Six: Isotopic labelings for mechanistic studiesIsotopic labelings for mechanistic studies

2 Investigations on the cyclization mechanisms of terpene synthases

2.1 Investigating reprotonations of neutral intermediates in terpene biosynthesis

2.1.1 Materials

2.1.2 Equipment

2.1.3 Before you begin

2.1.4 Procedure

2.1.5 Notes

2.2 Determination of the absolute configurations of terpenes through stereoselective deuteration

2.2.1 Materials

2.2.2 Equipment

2.2.3 Procedure

2.2.4 Notes

3 Conclusions and outlook

Chapter Seven: Understanding mechanisms of terpene synthases using substrate analogs

2 Overall strategy

Chapter Eight: Production of non-natural terpenoids through chemoenzymatic synthesis using substrate analogs

2 Equipment and reagents

3 Heterologous protein expression of PfCHK, MjIPK, (2E,6E)-GsFDPS and ScGDS Y406F

4 Purification of recombinant proteins PfCHK, MjIPK, (2E,6E)-GsFDPS and ScGDS Y406F

5 Chemical synthesis of non-natural analog of isoprenol (methyl-isoprenol)

6 Enzymatic incubations

6.1 Enzymatic incubations for synthesis of diphosphorylated prenol, isoprenol and methyl-isoprenol

6.2 Enzymatic incubations for synthesis of (S)-germacrene D (9) and (S)-14,15-dimethylgermacrene D (10).

6.3 Preparative-scale enzyme incubations for the preparation of (S)-germacrene D and (S)-14,15-dimethylgermacrene D

7 Issues related to substrate precipitation and product inhibition

8 Establishing reaction rate constants for promiscuous kinase activity

9 Summary

Acknowledgement

Chapter Nine: Docking carbocations into terpene synthase active sites using chemically meaningful constraints-The TerDockin approach

2 Methods

2.1 Optimization of terpene ligand using quantum mechanics

2.2 Conformer search and conformer library generation

2.3 Docking in Rosetta

2.3.1 Obtaining, and preprocessing the PDB file

2.3.2 Generating PARAMS files for ligands

2.3.3 Constraint file

2.3.4 Matching constraints to PDB header

2.3.5 Constraining the diphosphate for type I and type II terpene synthases

2.3.6 Flags file

2.3.7 XML file

2.3.8 Docking

3 Rosetta workup and analysis

4 Examples of research accomplished with TerDockin

4.1 Modeling of bornyl diphosphate synthase

4.2 Modeling and analysis of terpene synthase Rv3377c

4.3 Modeling and analysis of an ent-kaurene synthase from Bradyrhizobium japonicum

5 Concluding remarks

Chapter Ten: Mechanistic docking in terpene synthases using EnzyDock

2 Challenges in using standard docking tools for terpene synthases

3 Multistate multiscale docking with EnzyDock

3.1 CotB2-a toy system

3.2 The multistate concept

3.2.1 EnzyDock without restraints

3.2.2 EnzyDock with restraints

3.3 The multiscale concept

3.4 EnzyDock workflow

3.5 EnzyDock input

3.6 Practical aspects of EnzyDock

3.7 EnzyDock output and analysis

3.8 EnzyDock example

4 Concluding words

References.

Chapter Eleven: Bioinformatic analysis of microbial type terpene synthase genes in plants

2 Identification of putative microbial type terpene synthase genes from plants

2.1 Preparation of sequence database

2.2 Blast search

2.2.1 Blast search online

2.2.2 Local blast search on your computer

3 HMM search

3.1 Installing HMMER

3.2 Terpene HMM search profiles

3.3 HMM search

4 Validation of microbial type terpene synthase genes as plant genes

4.1 Domain analysis of hmmsearch results

4.2 Intron-exon structure analysis

4.3 Phylogenetic analysis

4.3.1 Multiple sequence alignment

4.3.2 Maximum likelihood tree reconstruction

4.3.2.1 Installing and conducting ProtTest3

Chapter Twelve: Ancestral terpene cyclases: From fundamental science to applications in biosynthesis

2 Ancestral sequence reconstruction as a protein engineering method

3 Ancestral sequence reconstruction of terpene synthases

3.1 Resources

3.2 Materials and equipment

3.4 Ancestral sequence reconstruction

3.5 Protein expression and purification (representative protocol for ancestral sesqui-/di- terpene cyclase)

3.6 Expected outcomes

3.6.1 Engineering of class I terpene synthases

3.6.2 Engineering of class II terpene synthases

3.6.3 Using ancestral sequence reconstruction as a protein engineering method of terpene synthases for applications in biosynthesis

3.6.4 Using ancestral sequence reconstruction to gain atomistic insight in catalysis by terpene synthases

3.7 Quantification and statistical analysis

3.8 Advantages

3.9 Limitations

3.10 Optimization and troubleshooting

3.10.1 Low bootstrap values

3.10.2 Potential solution to optimize the procedure

3.11 Safety considerations and standards

3.12 Alternative procedures.

4 Summary and conclusions.

Notes:

Includes bibliographical references.

Description based on publisher supplied metadata and other sources.

Description based on print version record.

ISBN:

0-443-23809-X

0-443-23808-1

OCLC:

1443938991