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Radical SAM enzymes /
"Radical SAM Enzymes, Volume 606, the latest release in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on the Characterization of the glycyl radical enzyme choline trimethylamine-lyase and its radical S-adenosylmethio...
| Clasificación: | Libro Electrónico |
|---|---|
| Otros Autores: | |
| Formato: | Electrónico eBook |
| Idioma: | Inglés |
| Publicado: |
Cambridge, MA :
Academic Press,
[2018]
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| Edición: | First edition. |
| Colección: | Methods in enzymology ;
v. 606. |
| Temas: | |
| Acceso en línea: | Texto completo |
Tabla de Contenidos:
- Front Cover; Radical SAM Enzymes; Copyright; Contents; Contributors; Preface; Chapter One: Atlas of the Radical SAM Superfamily: Divergent Evolution of Function Using a ``Plug and Play" Domain; 1. Introduction: Overview of the Radical SAM Superfamily; 2. Results and Discussion; 2.1. Structural Overview; 2.2. A New Classification of the RSS; 2.3. A Large-Scale View of Sequence-Structure-Function Relationships; 2.4. Ancient Lineage of the RSS; 2.5. Classification of the RSS Based on Sequence Similarity versus Chemical Similarity; 2.6. Targeting Unknowns for Experimental Characterization
- 3. Methods3.1. Collection of RSS Sequences; 3.2. Representative Networks; 3.3. Determining Subgroups and Families; 3.4. Annotation of the RSS in the SFLD; Acknowledgments; Author Contributions; Competing Financial Interests Statement; References; Chapter Two: Purification and Characterization of the Choline Trimethylamine-Lyase (CutC)-Activating Protein CutD; 1. Introduction; 2. Overproduction and Purification of CutD; 2.1. Gene Cloning and Overexpression Strategy; 2.2. Protocol for Overexpression of the CutD Gene; 2.3. Protocol for Purifying CutD Under Anaerobic Conditions
- 2.4. Procedure for Chemical Reconstitution of Isolated CutD3. Characterization of the Iron-Sulfur Clusters in CutD; 3.1. UV-vis Spectrum of Reconstituted CutD; 3.2. Protocol for EPR Analysis of Iron-Sulfur Clusters; 3.3. Generation of SAM Cleavage Products Monitored by HPLC; 4. Activity of CutD Toward CutC; 4.1. Protocol for Quantification of Extent of CutC Activation by EPR; 4.2. Protocol for Kinetic Analysis of Choline Cleavage by CutC Upon Activation by CutD; 4.3. Protocol for LC-MS/MS-Based Detection of TMA From CutC-Catalyzed Choline Cleavage; 5. Conclusions; Acknowledgments; References
- Chapter Three: QueE: A Radical SAM Enzyme Involved in the Biosynthesis of 7-Deazapurine Containing Natural Products1. Introduction; 2. Expression and Purification of QueE; 2.1. Expression of QueE; 2.2. Purification of QueE; 2.3. Chemical Reconstitution of QueE; 3. Expression and Purification of YkuN; 3.1. Expression of B. subtilis Flavodoxin YkuN; 3.2. Purification of YkuN; 4. Expression and Purification of FPR; 4.1. Expression of the fpr Gene; 4.2. Purification of FPR; 5. Enzymatic Synthesis of CPH4 With GCH I and QueD From GTP; 5.1. Expression and Purification of GCH I
- 5.2. Expression of QueD5.3. Purification of QueD; 5.4. Enzymatic Synthesis of CPH4; 6. Typical Assay Conditions for QueE; 6.1. Protocol for Assaying QueE In Vitro; 7. LC-MS Analysis of Turnover by CDG synthase; 8. Conclusions; Acknowledgments; References; Chapter Four: TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases; 1. Introduction; 2. Expression and Purification of TYW1; 2.1. Expression of TYW1 Gene; 2.1.1. Protocol for Expression of TYW1 Gene; 2.2. Purification of TYW1; 2.2.1. Protocol for Purification of His6-TYW1 Protein