Siderophore and Related Natural Products Biosynthesis / edited by Timothy Wencewicz.

Format:

Book

Contributor:

Wencewicz, Timothy, editor.

Series:

Methods in enzymology ; Volume 702.

Methods in Enzymology Series ; Volume 702

Language:

English

Subjects (All):

Siderophores.

Physical Description:

1 online resource (424 pages)

Edition:

First edition.

Place of Publication:

Cambridge, MA : Academic Press, [2024]

Summary:

This book provides an in-depth exploration of siderophores and related natural products, focusing on their biosynthesis, enzymology, and applications. It is part of the Methods in Enzymology series and includes detailed methodologies for the study of siderophore production, structure, and function. The book is edited by Timothy Wencewicz and includes contributions from experts in the field, offering comprehensive insights into the molecular biology and chemistry of siderophores. It is designed for researchers and professionals in biochemistry, molecular biology, and related fields, aiming to advance understanding of siderophore-mediated processes and their potential applications in medicine and biotechnology. Generated by AI.

Contents:

Front Cover

Series Page

Methods in Enzymology

Copyright

Contents

Contributors

Chapter One: Kinetic analysis of the three-substrate reaction mechanism of an NRPS-independent siderophore (NIS) synthetase

1 Introduction

2 Protein production and purification

2.1 Equipment

2.2 Buffers, strains, and reagents

2.3 Procedure

2.3.1 Protein expression

2.4 Notes

3 NADH coupled adenylation assay

3.1 Equipment

3.2 Reagents

3.3 Procedure

3.4 Notes

4 Kinetic analysis to distinguish among potential mechanisms

4.1 Possible mechanisms

4.2 Initial double reciprocal plots

4.3 Slope and intercept replots

4.4 Notes

5 Alternate approaches

6 Summary and conclusions

Acknowledgment

References

Chapter Two: Experimental methods for evaluating siderophore-antibiotic conjugates

2 Methods

2.1 General considerations for evaluation of SACs

2.2 Safety precautions and general practices

2.3 Antimicrobial activity (AMA) assay

2.3.1 Introduction

2.3.2 Materials

2.3.3 Procedure

2.4 Time-kill kinetics

2.4.1 Introduction

2.4.2 Materials

2.4.3 Procedure

2.5 Competition with unmodified Ent

2.5.1 Introduction

2.5.2 Materials

2.5.3 Procedure

2.6 57Fe uptake assay

2.6.1 Introduction

2.6.2 Materials

2.6.3 Procedure

3 Summary

Chapter Three: A continuous fluorescence assay to measure nicotianamine synthase activity

2 Assay design

3 Before you begin

4 Key resources table

5 Methods and equipment

5.1 Equipment

5.2 Reagents

6 Preparation of reagents

6.1 Purification of methylthioadenosine nucleosidase (MTAN)

6.2 Purification of adenine deaminase (AD)

6.3 Check for activity of MTAN and AD

7 Step-by-step method details

7.1 Prepare standard curve

7.2 Run the assay.

8 Quantification and statistical analysis

9 Summary

Chapter Four: ITC-based kinetics assay for NIS synthetases

2 General overview of the method

3.1 Protein preparation

4 Materials and equipment

5 Step-by-step method details

5.1 Optional recovery of siderophores

6 Expected outcomes

7 Quantification and statistical analysis

8 Optimization and troubleshooting

8.1 Problem: enzyme solubility

8.1.1 Exchange substrate and enzyme

8.1.2 Problem: V vs. [S] curves are not optimal

8.1.3 Increase [substrate] and/or [enzyme]

Chapter Five: An in vitro assay to explore condensation domain specificity from non-ribosomal peptide synthesis

2 General method and statistical analysis

3 Molecular design of PCP2-C3 SpyCatcher and PCP3 SpyTag constructs

3.3.1 Generating PCP2-C3 and PCP3 constructs

3.3.2 Generating PCP2-C3 SpyCatcher and PCP3 SpyTag constructs

4 Protein expression and purification

4.1 Reagents

4.2 Procedure

4.2.1 Transformation

4.2.2 Expression

4.2.3 Purification

4.2.3.1 Ni-NTA purification

4.2.4 PCP2-C3 SpyCatcher

4.2.5 Sfp mutant R4-4

4.2.5.1 Size exclusion chromatography

4.3 Notes

5 Synthesis of chemical reagents

5.3 Synthesis

5.3.1 Peptidyl-CoA

5.3.2 Aminoacyl-CoA synthesis

5.3.3 Stabilised aminoacyl-CoA

5.4 Notes

6 In vitro reconstitution of NRPS C-domain

6.1 Equipment

6.2 Reagents

6.3 Procedure

6.3.1 Loading of PCP-C with peptidyl-CoA

6.3.2 Reconstitution of NRPS peptide extension

6.4 Notes

7 LC-HRMS/MS analysis of methylamine cleaved peptide products

7.1 Material and equipment

7.2 Buffer and reagents

7.3 Procedures.

7.3.1 Methylamine cleavage of peptides from PCP domains

7.3.2 LC-HRMS/MS analysis of cleaved peptide products

7.4 Notes

8 Intact protein PPant ejection LC-ESI-Q-TOF-MS analysis of chemically stabilised peptide products

8.1 Equipment

8.2 Buffers and reagents

8.3 Procedure

8.4 Notes

9 Conclusions

Acknowledgements

Chapter Six: The production of siderophore analogues using precursor-directed biosynthesis

1.1 The clinical natural product desferrioxamine B

1.2 Structural diversification of desferrioxamine B using precursor-directed biosynthesis

1.3 Generation of constitutional isomers of desferrioxamine B analogues

1.4 Mass spectrometry to identify desferrioxamine B analogues and constitutional isomers

1.5 Scope of precursor-directed biosynthesis in producing desferrioxamine B analogues

1.6 Theoretical maxima of desferrioxamine B analogues produced using precursor-directed biosynthesis

2 Materials and equipment

2.1 Bacteria and chemicals

2.2 Consumables

2.3 General equipment

2.4 LC-MS-Q instrumentation

2.5 LC-MS/MS-QQQ instrumentation

3 Protocol

3.1 Before you begin

3.2 Preparing solutions and use of high grade reagents

3.3 Preparing frozen stocks of Streptomyces pilosus

3.4 Precursor-directed biosynthesis using non-native diamine substrates

3.5 Measuring siderophore production

3.6 Siderophore purification

3.7 Analysing desferrioxamine B analogues and constitutional isomers

4 Summary

Chapter Seven: Preparation of coenzyme F430 biosynthetic enzymes and intermediates

2 Expression and purification of the coenzyme F430 biosynthesis enzymes

2.1 HemC

2.2 HemD

2.3 SirA

2.4 SirC

2.5 CfbA

2.6 CfbB

2.7 CfbCD

2.8 CfbE

2.9 McrD.

3 Synthesis and purification of coenzyme F430 biosynthetic intermediates

3.1 Sirohydrochlorin

3.2 Ni-sirohydrochlorin

3.3 Ni-sirohydrochlorin a,c-diamide

3.4 15,173-seco-F430-173-acid

3.5 Coenzyme F430

4 Concluding remarks

Acknowledgments

Chapter Eight: Purification and biochemical characterization of methanobactin biosynthetic enzymes

2 Expression and purification of M. trichosporium OB3b MbnBC complexes

2.1 Plasmid construction and transformation into Escherichia coli

2.2 Large-scale growth and induction of MbnBC expression

2.3 Purification of MbnBC for biochemical studies

2.4 Purification of MbnBC for crystallization

3 In vitro modification of MbnA using purified MbnBC

4 Crystallization and structure determination of M. trichosporium OB3b MbnBC

5 Expression and purification of M. trichosporium OB3b MbnN

6 Conclusions

Chapter Nine: Discovery, isolation, and characterization of diazeniumdiolate siderophores

2 Bioinformatics and genome mining to predict bacteria producing graminine-containing siderophores

2.1 Materials

2.1.1 Equipment (software)

2.2 Constructing an SSN

2.3 Identifying bacterial strains of interest

3 Bacterial growth conditions for production of siderophores

3.1 Materials

3.1.1 Equipment

3.1.2 Reagents and supplies

3.2 Protocols for bacterial culturing

3.2.1 Preparation of iron-deficient media

3.2.2 Culture inoculation and monitoring

3.2.3 Measurement of optical density

4 Detection and isolation of the siderophores

4.1 Materials

4.1.1 Equipment

4.1.2 Reagents and supplies

4.2 CAS assay for general siderophore detection

4.2.1 Preparation of Fe-CAS-hexadecyltrimethylammonium bromide solution

4.2.2 CAS assay procedure.

4.3 Extraction of siderophores

4.3.1 Solid phase extraction of siderophores from bacterial culture

4.4 Purification of siderophores

4.4.1 Collecting fractions and identifying the siderophore HPLC peak

4.4.2 Lyophilization

5 Identification and characterization of graminine-containing siderophores

5.1 Materials

5.1.1 Equipment

5.1.2 Reagents and supplies

5.2 Mass spectrometry and stable isotope labeling

5.2.1 15N labeling

5.2.2 Deuterium (2H) labeling

5.2.3 Tandem MS/MS

5.3 Marfey's amino acid analysis

5.3.1 Hydrolysis of the siderophore

5.3.2 Derivatization of the hydrolysate and amino acid standards

5.3.3 Analytical HPLC

5.3.4 UPLC-MS

5.4 Photoreactivity of the diazeniumdiolate group

5.4.1 Photolysis of diazeniumdiolate siderophores

5.4.2 MS detection of NO mass loss

5.5 NMR spectroscopy characterization of the C-diazeniumdiolate and the photoproducts

6 Conclusions and future outlook

Chapter Ten: Linking biosynthetic genes to natural products using inverse stable isotopic labeling (InverSIL)

1.1 The gene-to-molecule approach for natural product discovery

1.2 Inverse stable isotopic labeling (InverSIL)

1.3 Using InverSIL to link quorum sensing signal synthase genes to their products

2 Key resources

3 Equipment and reagents

3.1 Inverse labeling of microbial culture

3.2 Natural product extraction

3.3 Analysis of labeled microbial extracts by untargeted mass spectrometry

3.4 Data analysis to identify inverse labeled natural products

4 Method

4.1 Inverse labeling of microbial culture

4.2 Natural product extraction

4.3 Analysis of labeled microbial extracts by untargeted mass spectrometry

4.4 Data analysis to identify inverse labeled natural products

5 Notes

References.

Chapter Eleven: 4-Aldrithiol-based photometric assay for detection of methylthioalkylmalate synthase activity.

Notes:

Includes bibliographical references.

Description based on publisher supplied metadata and other sources.

Part of the metadata in this record was created by AI, based on the text of the resource.

Description based on print version record.

ISBN:

9780443296796

0443296790

9780443296789

0443296782