This volume of "Methods in Enzymology" series is devoted to the cofactor thiamine, also known as vitamin B1, and its phosphorylated derivatives. The biosynthesis of thiamine is discussed in terms of key enzymes and their regulation, the evolution of metabolic pathways, and its importance in plant and yeast metabolism. The importance of quantifying and distinguishing between phosphorylated and unphosphorylated thiamine in animal and human tissues using chromatographic, enzymatic, and nanopore methods is also discussed. In addition to these methods, thiamine riboswitches are a major focus of this book, covering everything from their identification in plants and the elucidation of their structure to their application in detection.
This book presents state-of-the-art methods and detailed protocols intended for all researchers interested in thiamine metabolism or its detection.
1. Title of Book - p. i 2. Cover image - p. i 3. Title page - p. ii 4. Table of Contents - p. iii 5. Series Page - p. iii 6. Copyright - p. iv 7. Contributors - p. xvii 8. Preface - p. xxi 9. Section 1: Thiamine metabolism and assays - p. S1 10. Chapter One: Evolving thiazole synthases for expression in plants - p. 3 11. Chapter Two: Evolutionary reconstruction of thiamine biosynthesis pathway: An integrative bioinformatics workflow - p. 43 12. Chapter Three: Generation and characterization of thiamin-biofortified plants - p. 89 13. Chapter Four: Incubation and physiological readouts in vitro for thiamine triphosphate and related compounds in plants - p. 119 14. Chapter Five: ThiN as a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea - p. 149 15. Chapter Six: Assessment of thiamine status in humans - p. 189 16. Chapter Seven: Protocol for the measurement of human brain transketolase activity - p. 217 17. Chapter Eight: A general method for the assay of thiamine pyrophosphokinase activity - p. 239 18. Chapter Nine: Measurement of thiaminase I activity in biological samples: 4-nitrothiophenol (4-NTP) assays, optimization, and interpretation - p. 257 19. Chapter Ten: Engineered nanopore biosensor for thiamine quantification: Experimental design - p. 301 20. Chapter Eleven: Engineered nanopore biosensor for thiamine quantification: Data analysis - p. 327 21. Section 2: Riboswitches - p. S349 22. Chapter Twelve: Scaffold engineering enables high-resolution cryo-EM analysis of thiamine pyrophosphate riboswitch architecture and dynamics - p. 351 23. Chapter Thirteen: Targeting bacterial TPP riboswitches using pVEC-conjugated chimeric antisense oligonucleotides - p. 375 24. Chapter Fourteen: Characterization of metabolite-sensing riboswitches using in vivo reporter gene assays - p. 401 25. Chapter Fifteen: Mapping ligand-RNA interactions of thiamine analogues using transcriptome-wide sequencing - p. 437 26. Chapter Sixteen: Monitoring structural dynamics of nascent bacterial riboswitches responding to TPP within transcriptional complexes - p. 465
This volume of "Methods in Enzymology" series is devoted to the cofactor thiamine, also known as vitamin B1, and its phosphorylated derivatives. The biosynthesis of thiamine is discussed in terms of key enzymes and their regulation, the evolution of metabolic pathways, and its importance in plant and yeast metabolism. The importance of quantifying and distinguishing between phosphorylated and unphosphorylated thiamine in animal and human tissues using chromatographic, enzymatic, and nanopore methods is also discussed. In addition to these methods, thiamine riboswitches are a major focus of this book, covering everything from their identification in plants and the elucidation of their structure to their application in detection.
This book presents state-of-the-art methods and detailed protocols intended for all researchers interested in thiamine metabolism or its detection.
1. Title of Book - p. i 2. Cover image - p. i 3. Title page - p. ii 4. Table of Contents - p. iii 5. Series Page - p. iii 6. Copyright - p. iv 7. Contributors - p. xvii 8. Preface - p. xxi 9. Section 1: Thiamine metabolism and assays - p. S1 10. Chapter One: Evolving thiazole synthases for expression in plants - p. 3 11. Chapter Two: Evolutionary reconstruction of thiamine biosynthesis pathway: An integrative bioinformatics workflow - p. 43 12. Chapter Three: Generation and characterization of thiamin-biofortified plants - p. 89 13. Chapter Four: Incubation and physiological readouts in vitro for thiamine triphosphate and related compounds in plants - p. 119 14. Chapter Five: ThiN as a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea - p. 149 15. Chapter Six: Assessment of thiamine status in humans - p. 189 16. Chapter Seven: Protocol for the measurement of human brain transketolase activity - p. 217 17. Chapter Eight: A general method for the assay of thiamine pyrophosphokinase activity - p. 239 18. Chapter Nine: Measurement of thiaminase I activity in biological samples: 4-nitrothiophenol (4-NTP) assays, optimization, and interpretation - p. 257 19. Chapter Ten: Engineered nanopore biosensor for thiamine quantification: Experimental design - p. 301 20. Chapter Eleven: Engineered nanopore biosensor for thiamine quantification: Data analysis - p. 327 21. Section 2: Riboswitches - p. S349 22. Chapter Twelve: Scaffold engineering enables high-resolution cryo-EM analysis of thiamine pyrophosphate riboswitch architecture and dynamics - p. 351 23. Chapter Thirteen: Targeting bacterial TPP riboswitches using pVEC-conjugated chimeric antisense oligonucleotides - p. 375 24. Chapter Fourteen: Characterization of metabolite-sensing riboswitches using in vivo reporter gene assays - p. 401 25. Chapter Fifteen: Mapping ligand-RNA interactions of thiamine analogues using transcriptome-wide sequencing - p. 437 26. Chapter Sixteen: Monitoring structural dynamics of nascent bacterial riboswitches responding to TPP within transcriptional complexes - p. 465
