Methods in enzymology. Marine enzymes and specialized metabolism. Part B / editors in chief, Anna Marie Pyle and David W. Christianson.

Format:

Book

Contributor:

Pyle, Anna Marie, editor.

Christianson, David W., editor.

Language:

English

Subjects (All):

Enzymology.

Physical Description:

1 online resource (xvi, 549 pages)

Place of Publication:

Cambridge, Massachusetts : Academic Press, 2018.

Summary:

Marine enzymes and specialized metabolism - Part B, Volume 605 in the Methods in Enzymology series, highlights experimental methods on diverse marine enzymes involved in the construction of bioactive natural product molecules. Unique sections in this new release include discussions on polysaccharide-degrading enzymes from marine gastropods, radical SAM epimerases from sponge microbes, DMS/P demethylase in bacteria, reconstitution of particulate methane monooxygenase into membrane mimetics, the structure and function of cyanobactin enzymes, marine cyanobacterial polyketide beta-branching enzymology, marine cyanobacterial PKS-NRPS enzymology and structural biology, biochemical profiling of DMSP lyases, and more.- Subject not before covered in a methods book- Authority and expertise of the contributors

Contents:

Front Cover

Marine Enzymes and Specialized Metabolism Part B

Copyright

Contents

Contributors

Preface

Section I: Biosynthesis of Marine Lipids, Polyunsaturated Fatty Acids and Oxylipins

Chapter One: Characterization and Application of Marine Microbial Omega-3 Polyunsaturated Fatty Acid Synthesis

1. Introduction

2. Diversity and Distribution of Long-Chain Omega-3-Producing Marine Microbes

2.1. Marine Bacteria

2.2. Marine Labyrinthulomycetes and Other Microalgae

2.2.1. Protocol: Simple Enrichment and Isolation Strategy for Environmental Labyrinthulomycetes

3. The Pfa Synthase

3.1. Genetic and Catalytic Domain Architecture

3.2. Proposed Biosynthetic Process

4. Relationship Between LC-PUFA and LC-PUHC Biosynthesis

5. Cultivation and Genetic Optimization of Native Strains

5.1. Protocol: General Conjugation Protocol for Marine Bacteria

6. Recombinant Production of n-3 LC-PUFAs in Heterologous Hosts

7. Chemical Analysis Methods

7.1. Protocol: Whole-Cell Fatty Acid Methyl Esterification Derivatization Protocol

7.2. Protocol: Long-Chain Hydrocarbon Extraction Protocol

7.2.1. FAME GC-MS Method

7.2.2. PUHC GC-MS Method

8. Summary

Acknowledgments

References

Chapter Two: Expression of an 8R-Lipoxygenase From the Coral Plexaura homomalla

2. Preparation of Highly Purified 8R-LOX

2.1. Transformation of 8R-LOX psWT Into Overexpression Host

2.1.1. Equipment

2.1.2. Buffers and Reagents

2.1.3. Procedure

2.1.4. Notes

2.2. Expression of 8R-LOX

2.2.1. Equipment

2.2.2. Buffers and Reagents

2.2.3. Procedure

2.2.4. Notes

2.3. Purification of 8R-LOX

2.3.1. Equipment

2.3.2. Buffers and Reagents

2.3.3. Procedure

2.3.4. Notes

3. Structural Studies of 8R-LOX

3.1. Anaerobic Crystallization of 8R-LOX

3.1.1. Equipment.

3.1.2. Buffers and Reagents

3.1.3. Procedure (Carried Out Inside the Anaerobic Chamber)

3.1.4. Notes

3.2. Preparation of 8R-LOX:AA Binary Complexes

3.2.1. Equipment

3.2.2. Materials

3.2.3. Procedure (Carried Out Inside the Anaerobic Chamber)

3.2.4. Notes

4. Summary and Conclusions

Chapter Three: Catalase-Related Allene Oxide Synthase, on a Biosynthetic Route to Fatty Acid Cyclopentenones: Expression ...

Abbreviations

1.1. Nomenclature and Occurrence

1.2. cAOS-LOX Fusion Proteins

2. Preparation of P. homomalla cAOS Enzyme

2.1. Overview

2.2. Bacterial Expression and Initial Isolation Using Ni-NTA

2.3. Nickel Affinity Chromatography

2.4. Purification by Anion-Exchange Chromatography

2.5. UV Assay of cAOS and HPLC of Products

3. Preparation of 8R-HPETE: Method 1, via Autoxidation of Arachidonate

3.1. Overview, Vitamin E-Controlled Autoxidation

3.2. Vitamin E-Controlled Autoxidation, Example

3.3. Chromatographic Separation of HPETE Isomers

3.4. Hydrolysis of HPETE-Me to the Free Acid

3.5. Chiral Resolution of 8RS-HPETE

4. Preparation of 8R-HPETE: Method 2, Using 8R-LOX

4.1. Substrate Solubility Issues

4.2. Pretesting 8R-LOX for Activity

4.3. Preparative Reaction

4.4. Extraction and Purification

5. Key Differences Between HPETE-Metabolizing cAOS and Classic Catalases

5.1. Substrate Access to the Distal Heme

5.2. Val/Thr Substitution on the Distal Heme

Chapter Four: Lipoxygenases and Lipoxygenase Products in Marine Diatoms

2. Lipoxygenases and Enzymes of the LOX Pathways in Diatoms

3. Biosynthesis of Diatom Oxylipins

4. Analytical Methods

4.1. Activation of Lipoxygenase Pathway and Sample Extraction

4.1.1. Equipment.

4.1.2. Solvents and Reagents

4.1.3. Procedure

4.1.4. Notes

4.2. LC-MS/MS Analysis of NVOs and Identification of Lipoxygenases

4.2.1. Equipment

4.2.2. Solvents and Reagents

4.2.3. Sample Preparation

4.2.4. Chromatographic and MS Settings for Micro-qToF Analysis

4.2.5. Chromatographic and MS Settings for Q-Exactive Orbitrap Analysis

4.3. GC-MS Analysis of Hydroxy Fatty Acids

4.3.1. Equipment

4.3.2. Reagents

4.3.3. Procedure

4.4. GC-MS Analysis of PUAs

4.4.1. Equipment

4.4.2. Reagents

4.4.3. Procedure

4.4.4. Notes

5. Analysis of NVOs in Marine Phytoplankton

6. Conclusions

Chapter Five: Sterol Sulfates and Sulfotransferases in Marine Diatoms

2. Sulfotransferases

3. SULTs and Sterol Sulfates in Marine Diatoms

4. Methods

4.1. Isolation and Purification of Sterol Sulfates

4.1.1. Algal Culture and Sample Preparation

4.1.1.1. Equipment

4.1.1.2. Buffers and Reagents

4.1.1.3. Procedure

4.1.1.4. Notes

4.1.2. Solid-Phase Extraction

4.1.2.1. Equipment

4.1.2.2. Solvents and Reagents

4.1.2.3. Procedure

4.1.2.4. Notes

4.1.3. HPLC Purification

4.1.3.1. Equipment

4.1.3.2. Solvents and Reagents

4.1.3.3. Procedure

4.2. Identification of Sterol Sulfates

4.2.1. Chemical Conversion of Sterols to Sterol Sulfates

4.2.1.1. Equipment

4.2.1.2. Solvents and Reagents

4.2.1.3. Procedure

4.2.2. NMR Analysis

4.2.2.1. Equipment

4.2.2.2. Solvents and Reagents

4.2.2.3. Procedure

4.2.2.4. Notes

4.3. UPLC-MS Analysis of Sterol Sulfates

4.3.2. Solvents and Reagents

4.3.4. Notes

4.4. Inhibition of Sterol Sulfate Biosynthesis in Marine Diatoms

4.4.2. Buffers and Reagents

4.4.4. Notes.

4.5. 13C Labeling of the Carbon Skeleton of Diatom Sterols

4.5.1. Equipment

4.5.2. Buffers and Reagents

4.5.3. Procedure

4.5.4. Notes

5. Conclusions

Section II: Marine Halogenating and Dehalogenating Enzymes

Chapter Six: Marine Vanadium-Dependent Haloperoxidases, Their Isolation, Characterization, and Application

2. Presence of VHPOs in Marine Algae and Bacteria

2.1. VHPOs in Seaweeds

2.2. Peroxidases in Phytoplankton

2.3. VHPOs in Bacteria

2.3.1. Cyanobacteria

2.3.2. Marine Streptomycetes

2.3.3. Flavobacteria

2.4. Activity Assays for IPO and BPO Activity

2.4.1. IPO Activity

2.4.2. BPO Activity

2.4.2.1. Phenol Red Assay

2.4.2.2. Monochlorodimedone Assay

2.4.2.3. Notes

2.5. Apoenzyme and Reconstitution of Activity

2.5.1. Procedure for Preparing Apo and Holoenzyme

2.5.1.1. Notes

2.6. Stability of VHPOs During Turnover

3. Isolation Procedures of Iodo- and BPOs From Seaweeds

3.1. Isolation of Vanadium-Dependent BPOs From Brown Seaweeds

3.1.1. Equipment

3.1.2. Buffers and Reagents

3.1.3. Procedure

3.2. Isolation of A. nodosum VBPO II From the Thallus Surface

3.2.2. Buffers and Reagents

3.3. Isolation of Iodo- and BPOs From the Laminariaceae Family Using a Two-Phase System

3.3.1. Procedure

3.3.2. Notes

4. Expression of Recombinant Bromo- and IPOs in Convenient Hosts

4.1. BPOs From Brown Seaweed

4.2. BPO From Various Red Seaweeds

4.3. Bacterial IPO and BPO

5. Choosing Correct Assay Conditions: Kinetic Considerations and Specificity

5.1. Assay Conditions to Assess Brominating Activity of Seaweed Enzymes

5.1.1. Notes

5.2. Assay Conditions to Assess Brominating Activity of CPOs From Dematiaceous Hyphomycetes

5.2.1. Note.

5.3. Assay Conditions to Assess Chlorinating Activity of CPOs From Dematiaceous Hyphomycetes

5.3.1. Notes

5.4. Kinetic Parameters for the Oxidation of Halides

6. Structural Aspects

6.1. SDS Gel Electrophoresis

6.1.1. Reagents

6.1.2. Procedure

7. Spectroscopic Methods and Biophysical Techniques to Study Active Sites of VHPOs

7.1. UV-Vis

7.1.1. Notes

7.2. EPR on VHPOs

7.3. Magic Angle Spinning 51V Solid-State NMR

7.3.1. Note

7.4. X-ray Crystallography of VHPOs

7.4.1. Notes

7.5. V-XAS and EXAFS

7.5.1. Notes

8. Structural Homology of Marine Iodo- and BPOs, Terrestrial Vanadium-Dependent CPOs, and Acid Phosphatases

8.1. Note

9. Potential Applications of VHPOs

9.1. Synthesis of Halogenated Compounds by VHPOs

9.1.1. Selectivity

9.2. Sulfoxidation by Vanadium-Dependent BPOs

9.2.1. Notes

9.3. Singlet Oxygen Production

9.3.1. Notes

9.4. Selective Oxidative Carboxylation of Amino Acids

9.5. Cascades

10. Conclusions

Chapter Seven: Haloalkane Dehalogenases From Marine Organisms

2. Identification of HLDs From Marine Organisms

2.1. HLDs From Pollution-Degrading Microorganisms

2.2. HLDs From Symbiotic Microorganisms

2.3. HLDs From Genomic Databases

2.4. HLDs From Metagenomic Libraries

3. Characteristics of Marine HLDs

3.1. Specific Activity

3.2. Steady-State Kinetics

3.3. Enantioselectivity

3.4. Structure and Stability

3.4.1. Analysis of Crystal Structures

3.4.2. Quaternary Structure

3.4.3. Effects of pH, Temperature, and Solvents on Conformational Stability

4. Experimental Characterization of HLDs

4.1. Expression and Purification

4.2. Specific Activity

4.2.1. Colorimetric Assay

4.2.1.1. Principle

4.2.1.2. Chemicals

4.2.1.3. Material and Equipment

4.2.1.4. Calibration.

4.2.1.5. Procedure.

Notes:

Description based on print version record.

ISBN:

0-12-815046-7

0-12-815045-9