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This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. The second of 3 volumes coveri… Read more
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Chapter One. In Vivo Production of Thiopeptide Variants
1 Introduction
2 Generation of Thiopeptide Variants
References
Chapter Two. Microviridin Biosynthesis
1 Introduction
2 Reconstitution In Vitro of the Cyclization Reactions of the Microviridin Biosynthetic Pathway from P. agardhii
3 Influence of Leader Peptide on the Cyclization Reactions
4 Summary
References
Chapter Three. Cyclotide Isolation and Characterization
1 Introduction
2 Isolation of Cyclotides from Plant Tissues
3 Isolation of Nucleic Acids Encoding Cyclotide Precursors
4 Mass Spectrometric Detection and Characterization of Cyclotides
5 Structural Analysis of Cyclotides
6 Membranolytic Assays of Cyclotides
7 Summary
References
Chapter Four. Ribosomally Encoded Cyclic Peptide Toxins from Mushrooms
1 Introduction
2 Detection and Purification of Amanita Cyclic Peptide Toxins
3 Identification of Cyclic Peptide Genes in Amanita and Other Fungi
4 Purification and Assay of Prolyl Oligopeptidase (POP) from Mushrooms
5 Immunodetection of POP in Mushroom Tissues
6 Summary
References
Chapter Five. The Pictet–Spengler Mechanism Involved in the Biosynthesis of Tetrahydroisoquinoline Antitumor Antibiotics
1 Introduction
2 Biosynthetic Analysis of THIQ Core Scaffold Assembly
3 Reaction Mechanism for Constructing a Pentacyclic THIQ Core Skeleton Catalyzed by a Novel Function for a Single NRPS Module
4 Comparison Between Typical Peptide Synthesis and the Pictet–Spengler Reaction Catalyzed by NRPS Machinery
5 Reaction Profiles Affected by the Chain Length of the Acyl Moiety
6 Multiple Roles for the Fatty Acyl Chain During the Maturation Process in Late-Stage Biosynthesis
7 Methods for Substrate Synthesis, Enzyme Preparation, and Assay Conditions
8 Conclusion
References
Chapter Six. Discovery and Biosynthesis of Phosphonate and Phosphinate Natural Products
1 Introduction
2 Spectrum of Phosphonate Natural Products: Structures and Bioactivities
3 Biosynthetic Pathways
4 Screening Bacteria for Phosphonate Producing Gene Clusters
5 Purification of Phosphonates/Phosphinates
6 Structural Elucidation of Phosphonates
7 A Wealth of Unusual Biosynthetic Enzymes
8 Conclusion and Outlook
References
Chapter Seven. RlmN and AtsB as Models for the Overproduction and Characterization of Radical SAM Proteins
1 Introduction
2 RlmN as a Model for Gene Expression and Overproduction of RS Proteins
3 RlmN as a Model for Purification of RS Proteins
4 Overproduction and Purification of E. coli Flavodoxin and Flavodoxin Reductase
5 AtsB as a Model for Determination of Configuration and Stoichiometry of Iron–Sulfur Clusters in RS Enzymes
6 Activity Determination of RS Enzymes
References
Chapter Eight. Fe(II)-Dependent, Uridine-5′-Monophosphate α-Ketoglutarate Dioxygenases in the Synthesis of 5′-Modified Nucleosides
1 Introduction
2 Methods
3 Summary
References
Chapter Nine. Tailoring Reactions Catalyzed by Heme-Dependent Enzymes
1 Introduction
2 Overexpression, Purification, and Characterization of the Novel Nitrating CYP TxtE
3 Protein Characterization Using UV–vis Spectroscopy
4 Ligand and Substrate Binding
5 Summary
References
Chapter Ten. Oxidative Tailoring Reactions Catalyzed by Nonheme Iron-Dependent Enzymes
1 Introduction
2 Examples of Tailoring Reactions Catalyzed by Nonheme Iron- and Oxygen-Dependent Enzymes
3 Heterologous Expression and Functional Investigation of the S. coelicolor redH and redG Genes in Streptomyces albus
4 Conclusions
References
Chapter Eleven. The Rare Fluorinated Natural Products and Biotechnological Prospects for Fluorine Enzymology
1 Introduction
2 Production of Fluoroacetate and 4-Fluorothreonine from S. cattleya
3 Enzyme 1: Fluorinase
4 Enzyme 2: Purine Nucleoside Phosphorylase
5 Enzyme 3: Isomerase
6 Enzyme 4: Aldolase
7 Enzyme 5: Transaldolase (Pyridoxal Phosphate)
8 Enzyme 6: Aldehyde Dehydrogenase
9 Generation of a Novel Fluorometabolite by Genetic Engineering
10 Summary
References
Chapter Twelve. Enzymatic Chlorination and Bromination
1 Introduction
2 FADH2-Dependent Halogenases
3 Characterization of FADH2-Dependent Halogenases
4 Nonheme Iron, α-Ketoglutarate- and O2-Dependent Halogenases
References
Chapter Thirteen. Prenyltransferases of the Dimethylallyltryptophan Synthase Superfamily
1 Introduction
2 Spore Preparation
3 Cultivation of Fungi
4 RNA and DNA Isolation and cDNA Synthesis
5 Gene Cloning
6 Protein Overproduction
7 Preparation of Stock Solutions for Enzyme Assays
8 Biochemical Characterization of Prenyltransferases
9 Chemoenzymatic Synthesis of Prenylated Compounds
10 HPLC
11 Summary
References
Chapter Fourteen. Serine Carboxypeptidase-Like Acyltransferases from Plants
1 Introduction
2 Methods in SCPL Acyltransferase Research
3 Summary
References
Chapter Fifteen. 4-Methylideneimidazole-5-One-Containing Aminomutases in Enediyne Biosynthesis
1 Introduction
2 Methods
3 Conclusion
References
Chapter Sixteen. Tailoring Enzymes Acting on Carrier Protein-Tethered Substrates in Natural Product Biosynthesis
1 Introduction
2 Methods
3 Conclusion
References
Chapter Seventeen. High-Throughput Colorimetric Assays for Nucleotide Sugar Formation and Glycosyl Transfer
1 Introduction
2 Purification of the Glycosyltransferase OleD
3 Colorimetric Assay for NDP-Sugar Formation
4 Coupled Reactions for Glycosyl Transfer
5 High-Throughput Colorimetric Coupled Assay for Glycosyl Transfer
6 Summary
References
Author Index
Subject Index
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