Mononuclear Non-Heme Iron Dependent Enzymes / Anna Maria Pyle, David Christianson, and Jennifer Bridwell-Rabb.

Format:

Book

Author/Creator:

Pyle, Anna Maria, author.

Christianson, David, author.

Bridwell-Rabb, Jennifer, author.

Series:

Issn Series

Language:

English

Subjects (All):

Biocatalysis.

Biodegradation.

Nitroaromatic compounds.

Physical Description:

1 online resource (348 pages)

Edition:

First edition.

Place of Publication:

Cambridge, MA : Academic Press, [2024]

Summary:

Mononuclear Non-heme Iron Dependent Enzymes, Volume 703 focuses on methods for studying, characterizing, and leveraging the chemistry of mononuclear non-heme iron dependent enzymes. Chapters in this new release include Photoreduction for Rieske oxygenase chemistry, Insights into the Mechanisms of Rieske Oxygenases from Studying the Unproductive Activation of Dioxygen, Non-heme iron and 2-oxoglutarate enzymes catalyze cyclopropane and azacyclopropane formations, Obtaining precise metrics of substrate positioning in Fe(II)/2OG dependent enzymes using Hyperfine Sublevel Correlation Spectroscopy, Xe-pressurization studies for revealing substrate-entrance tunnels, and much more.Additional chapters cover A tale of two dehydrogenases involved in NADH recycling, Rieske oxygenases and/or their partner reductase proteins, Expression, assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays, Biocatalysis and non-heme iron enzymes, In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01, Structure and function of carbazole 1,9a-dioxygenase, Characterization of a Mononuclear Nonheme Iron-dependent Mono-oxygenase OzmD in Oxazinomycin Biosynthesis, and much more.- Provides detailed articles regarding how to study the structures and mechanisms of mononuclear non-heme iron dependent enzymes- Guides readers on how to use partner proteins in non-heme iron enzyme catalysis- Includes strategies to employ mononuclear non-heme iron enzymes in biocatalytic applications

Contents:

Front Cover

Series Page

Methods in Enzymology

Copyright

Contents

Contributors

Section 1: Methods for studying the catalytic mechanisms of mononuclear non-heme iron dependent enzymes

Chapter One: Characterization of O2 uncoupling in biodegradation reactions of nitroaromatic contaminants catalyzed by rieske oxygenases

1 Introduction

2 Mass Balances and Reaction Stoichiometries

2.1 Quantification strategies and limitations

2.2 Quantification of O2 uncoupling

2.2.1 Experimental design

2.2.2 Equipment

2.2.3 Procedure for controlled substrate turnover experiments

2.2.4 Blank experiments

2.2.5 Quantification of O2 uncoupling and background O2 consumption

2.2.6 Procedure for quantification of O2 uncoupling

3 Quantification of transient reactive oxygen species

3.1 Survey of ROS quantification approaches

3.2 Catalase-based assays

3.2.1 Equipment

3.2.2 Procedure

3.3 Horseradish peroxidase-based assays

3.3.1 Equipment

3.3.2 Procedure

4 Reaction kinetics

4.1 Quantification of O2 uncoupling from reaction kinetics

4.2 Organic substrate kinetics

4.2.1 Procedure

4.3 Oxygen consumption kinetics

4.3.1 Procedure

5 Summary and Conclusions

Acknowledgments

Competing interests

References

Chapter Two: Spectroscopic definition of ferrous active sites in non-heme iron enzymes

1 d6 Ligand field theory (LFT)

2 LF spectroscopy = low temperature magnetic circular dichroism (LT MCD)

3 Variable-temperature, variable-field (VTVH) MCD

4 LFT of spin-hamiltonian parameters from VTVH MCD

5 An early application of VTVH MCD on a non-heme Fe(II) enzyme

6 Perspective

Chapter Three: Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase

2 Materials.

2.1 Quantitation of ligand and calibration curve

2.2 Equilibrium dialysis apparatus setup and initial measurements

2.3 Equilibrium dialysis for determination of substrate affinity in BesD

3 Methods

3.1 Quantitation of ligand and calibration curve

3.2 Equilibrium dialysis apparatus setup and initial measurements

3.3 Equilibrium dialysis for determination of substrate affinity in BesD

4 Conclusions

Author contributions

Chapter Four: Preparation of reductases for multicomponent oxygenases

2 General safety

3 Reductase production and activity

3.1 Overview

3.2 Expression vector cloning

3.2.1 Equipment and materials

3.3 Transformation of E. coli and RHA1 with expression vectors

3.3.1 Equipment and materials

3.4 Production of PbdB

3.4.1 Equipment and materials

3.4.2 Procedure

3.5 Activity of lysates

3.5.1 Equipment and materials

3.5.2 Procedure

3.6 Results

4 Codon optimization and protein purification

4.1 Overview

4.2 Codon optimization

4.2.1 Equipment and materials

4.2.2 Procedure

4.3 Protein purification

4.3.1 Equipment and materials

4.3.2 Procedure

4.4 Results

5 Protein characterization

5.1 Overview

5.2 Cofactor analysis - labile sulfide

5.2.1 Equipment and materials

5.2.2 Procedure

5.3 Cofactor analysis - non-heme iron

5.3.1 Equipment and materials

5.3.2 Procedure

5.4 Cofactor analysis - FAD

5.4.1 Equipment and materials

5.4.2 Procedure

5.5 Activity analysis - cytochrome c reduction

5.5.1 Equipment and materials

5.5.2 Procedure

5.6 Results

6 Summary and conclusions

Chapter Five: Development of a rapid mass spectrometric method for the analysis of ten-eleven translocation enzymes

1 Introduction.

2 Preparation of the materials

2.1 Expression and purification of wild type TET2

2.1.1 Materials and reagents

2.1.2 Equipment

2.1.3 Protocol

2.2 Mutagenesis and expression of V1395A

2.2.1 Materials and reagents

2.2.3 Protocol

2.3 Synthesis and characterization of oligonucleotides

2.3.1 Materials and reagents

2.3.2 Equipment

2.3.3 Protocol

3 Biochemical assays and results

3.1 Development of a robust in-vitro assay

3.1.1 Materials and reagents

3.1.2 Equipment

3.1.3 Protocol

3.2 Measurement of IC50 of TET2 inhibitors NOG and 2HG

3.2.1 Materials and reagents

3.2.2 Equipment

3.2.3 Protocol

3.3 Validating the activity of wildtype TET2 and V1395A using BS-seq

3.3.1 Materials and reagents

3.3.2 Equipment

3.3.3 Protocol

4 Notes

Funding

Chapter Six: Non-standard amino acid incorporation into thiol dioxygenases

1 Overview

2 Eukaryotic thiol dioxygenases

2.1 CDO

2.2 ADO

3 Genetic code expansion

3.2 Suppressor tRNA/aminoacyl‑tRNA synthetase pairs and the pEVOL plasmid

3.3 Selenocysteine incorporation

4 Application to thiol dioxygenases

4.1 Fluorotyrosine incorporation into CDO and ADO using pEVOL F2Y

4.2 Sec incorporation into ADO

5 Conclusions

Further reading

Chapter Seven: Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach

2 Protein expression, purification, and crystallization

2.1 Equipment

2.2 Reagents

2.3 Procedure

2.4 Note

3 Spectral characterization of Co-ADO

3.1 Equipment

3.2 Reagents

3.3 Optical spectral characterization

3.4 EPR spectral characterization

4 Cobalt reconstitution in ADO

4.1 Equipment

4.2 Reagents.

4.3 Preparation of "apo-ADO" through 1,10-phenanthroline assay

4.4 Evaluation of the "apo-ADO" using ferrozine assay

4.5 Reconstitution of ADO enzyme by adding divalent metal ions

4.6 Note

5 HPLC-MS analysis of the hypotaurine formation by ADO

5.1 Equipment

5.2 Reagents

5.3 Procedure

5.4 Note

Chapter Eight: In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01

2 In vitro analysis of Rieske oxygenases

3 Expression of Cumene dioxygenase

3.1 Materials

3.1.1 Strains

3.2 Buffers and reagents

3.3 Equipment

4 Step-by-step method details

5 General considerations

6 Cell lysis and protein purification

6.1 Materials and equipment

6.1.1 Buffers and reagents

6.2 Equipment

7 Step-by-step method details

7.1 Cell lysis by sonication

7.2 Purification by immobilized metal ion chromatography (IMAC)

7.3 Desalting by size exclusion chromatography (SEC)

7.4 Concentration by centrifugal ultrafiltration

8 General considerations

9 Enzymatic activity assay

9.1 Materials and equipment

9.1.1 Buffers and reagents

9.2 Equipment

10 Step-by-step method details

10.1 Enzymatic reaction

10.2 Sample extraction

10.3 Non-chiral GC-MS and chiral GC-FID analysis

11 General considerations

12 Summary and conclusions

Section 2: Leveraging mononuclear non-heme iron enzymes for biocatalysis

Chapter Nine: Radical-relay C(sp3)-H azidation catalyzed by an engineered nonheme iron enzyme

2 Materials

2.1 Cloning

2.2 Enzyme expression in E. coli

2.3 Whole-cell reaction

2.4 GCMS (gas chromatography-mass spectrometry) and normal phase HPLC (high performance liquid chromatography) analysis.

3 Protocols

3.1 Cloning for a site-saturated mutagenesis screening library

3.2 High-throughput experimentation in 96-well plates

3.3 Analytical scale reactions to validate the screening hits

3.4 Preparative-scale reactions

4 Summary

Chapter Ten: Purification and characterization of a Rieske oxygenase and its NADH-regenerating partner proteins

2 Considerations for assembling a Rieske oxygenase pathway in vitro

3 Protein constructs for recombinant expression and purification

3.1 Assembly of needed constructs for protein isolation

3.2 Transformation protocol for the TsaMBCD pathway encoding genes

4 Recombinant expression and purification of the TsaM, TsaC, TsaD, and VanB

4.1 Recombinant expression of the TsaM, TsaC, TsaD, and VanB encoding genes

4.2 Purification of TsaM, VanB, and TsaC

4.3 Purification of the NAD+-dependent aldehyde dehydrogenase TsaD

5 Methods for assessing the quality of the purified TsaMBCD pathway proteins

5.1 Biochemical analysis of purified proteins

5.2 Quantification of the iron content in TsaM and VanB

6 Enzymatic assays for the TsaMBCD pathway

6.1 Liquid chromatography mass spectrometry (LC-MS) methods for activity assays

6.2 Separation of TsaMBCD pathway intermediates using LC-MS

6.3 Identification of the optimal conditions for measuring the activity of TsaM

6.4 Total turnover number (TTN) determination using LC-MS

6.5 Spectroscopic assay for analysis of NAD(P)H consumption and production

7 Crystallization of the short-chain dehydrogenase/reductase (SDR) enzyme TsaC

8 Conclusions

Chapter Eleven: Whole-cell Rieske non-heme iron biocatalysts

2 Before you begin timing: 4-5 days

3 Key resources table

4 Materials and equipment

4.1 Equipment.

4.2 Materials and reagents.

Notes:

Includes bibliographical references.

Description based on publisher supplied metadata and other sources.

Description based on print version record.

ISBN:

0-443-31305-9

0-443-31304-0