P450 Enzymes

1st Edition, Volume 693 - November 13, 2023
Editor: Hans Renata
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 1 3 6 9 5 - 5
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 1 3 6 9 6 - 2

P450 Enzymes, Volume 692 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters written by an international… Read more

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P450 Enzymes, Volume 692 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters written by an international board of authors. Chapters in this new release cover Non-natural P450 reaction (carbene/nitrene), Non-natural P450 reaction (atom transfer radical cyclization), P450 fusion for biocatalysis, P450 fingerprinting for biocatalytic oxidation, Fungal peroxygenase, Fungal peroxygenase, Anti-Markovnikov P450 oxygenase (AmOX), Fungal P450, Unusual P450 chemistry from fungal sources, CASTing for P450 biocatalysis, and P450 in C–C coupling of cyclic dipeptides with nucleobases.

Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Preface
Chapter One: Directed evolution of P411 enzymes for amination of inert C–H bonds
Abstract
1 Introduction
2 Generation of mutant libraries
3 Protein expression
4 Enzymatic reaction setup
5 Screening and validation of the enzymatic reactions
6 Summary
References
Chapter Two: P450-catalyzed atom transfer radical cyclization
Abstract
1 Introduction
2 Materials
3 Protocols
4 Summary
Acknowledgments
References
Chapter Three: Self-sufficient P450-reductase chimeras for biocatalysis
Abstract
1 Introduction
2 Protocols
3 Biocatalytic reactions with PtmO5-RhFRed
4 Developing TleB as a self-sufficient P450 biocatalyst
5 Biocatalytic reaction with TleB-RhFRed
Acknowledgments
References
Chapter Four: The colors of peroxygenase activity: Colorimetric high-throughput screening assays for directed evolution
Abstract
1 Introduction
2 Materials, equipment and reagents
3 Enzyme production
4 Validation of the assay
5 High-throughput screening (HTS) assays
6 Discussion and outlook
References
Chapter Five: Engineering cytochrome P450s for selective alkene to carbonyl oxidation
Abstract
1 Introduction
2 General methods
3 Library construction
4 Recombinant protein expression in 96-well plate format
5 Library screening
6 Rescreening of variants containing beneficial mutations
7 Summary and conclusions
Acknowledgment
References
Chapter Six: Bacterial cytochrome P450 enzymes: Semi-rational design and screening of mutant libraries in recombinant Escherichia coli cells
Abstract
1 Introduction
2 Cloning of surrogate redox partners for CYP107Z
3 Construction of focused P450 mutant library
4 Expression of site-saturation libraries and whole-cell biotransformation of (−)-deoxypodophyllotoxin in 96-deep-well plates
5 MISER LC/MS analysis
6 Verification of selected P450 variants for in vitro conversion of (−)-deoxypodophyllotoxin
7 Summary and conclusion
References
Chapter Seven: Functional analysis of a fungal P450 enzyme
Abstract
1 Introduction
2 In vivo analysis of Trt6 and Trt14
3 In vitro analyses of Trt6 and Trt14
Acknowledgments
References
Chapter Eight: Protein engineering using mutability landscapes: Controlling site-selectivity of P450-catalyzed steroid hydroxylation
Abstract
1 Introduction
2 Mutability landscapes
3 Identification of hotspots by mutability landscape and empirical mutations
4 Enzyme engineering by iterative saturation mutation (ISM) and rational design
5 Exploration of the mutants to hydroxylate other steroids
6 Materials, equipment and reagents
7 Protocol
8 Summary
Acknowledgments
References
Chapter Nine: P450 in C–C coupling of cyclodipeptides with nucleobases
Abstract
1 Introduction
2 General instruction and cultivation of bacterial strains
3 Genomic DNA isolation
4 PCR amplification for P450 gene cloning
5 Cloning of plasmids and transformation of E. coli
6 Heterologous gene expression in Streptomyces
7 Protein overproduction in E. coli and purification
8 SDS-PAGE
9 Enzyme assays
10 Product isolation and structure elucidation
11 Summary and conclusions
Acknowledgments
References
Chapter Ten: Secretion and directed evolution of unspecific peroxygenases in S. cerevisiae
Abstract
1 Introduction
2 Generation of a signal peptide library
3 Library cultivation
4 Colorimetric screening methods
5 Isolation of episomal plasmids from S. cerevisiae
6 Golden Mutagenesis
7 Performing reactions in 96-well microtiter plate
8 MISER-GC–MS
9 Summary and conclusion
Acknowledgment
References
Chapter Eleven: Direct aromatic nitration by bacterial P450 enzymes
Abstract
1 Introduction
2 Creation of self-sufficient TxtE chimeras
3 Spectral analysis of TxtE fusion variants
4 Evaluation of TxtEBM3R chimera nitration activity
5 Summary and conclusions
Acknowledgments
References
Chapter Twelve: P450 fatty acid decarboxylase
Abstract
1 Introduction
2 Discovery of P450 fatty acid decarboxylases
3 Expression, characterization and structure analysis of P450 fatty acid decarboxylases
4 Engineering of P450 fatty acid decarboxylases
5 Applications of P450 fatty acid decarboxylases
6 Conclusion
Acknowledgments
References
Chapter Thirteen: Directed evolution for Si–C bond cleavage of volatile siloxanes in glass bioreactors
Abstract
1 Introduction
2 Generation and characterization of enzyme libraries
3 Assembling the components for reactions and reaction analysis with the glass bioreactors
4 Screening enzyme mutant libraries in glass 96-well bioreactors
5 Modification of procedures for validating improved enzyme variants
6 Summary
Acknowledgements
References

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Hans Renata