
Coenzyme B12 Enzymes Part A
- 1st Edition, Volume 668 - May 17, 2022
- Imprint: Academic Press
- Editor: Neil Marsh
- Language: English
- Hardback ISBN:9 7 8 - 0 - 3 2 3 - 9 1 5 9 1 - 5
- eBook ISBN:9 7 8 - 0 - 3 2 3 - 9 1 5 9 2 - 2
B12 Enzyme, Volume 667 in the Methods in Enzymology serial, highlights new advances in the field with this new volume presenting timely, interesting chapters, including Par… Read more

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Request a sales quoteB12 Enzyme, Volume 667 in the Methods in Enzymology serial, highlights new advances in the field with this new volume presenting timely, interesting chapters, including Part I: B12 Biosynthesis and B12 Chaperones, Probing the preference and regioselectivity of lower ligand activation and its role in cobamide diversity, Biosynthesis of corrinoids, The human B12 trafficking chaperones: CblC, CblD and ATR, A method for the efficient adenosylation of corrinoids, Production, purification and liposome reconstitution of cobamide synthase, Extraction and Purification of Cobamides from Bacterial Cultures, Part II: The many faces of B12-dependent enzyme reactions, Methylcobalamin-dependent methionine synthase, and much more.
- Provides the authority and expertise of leading contributors from an international board of authors
- Presents the latest release in Methods in Enzymology serials
- Includes the latest information on B12 Enzymes
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
- Cover
- Title page
- Table of Contents
- Copyright
- Contributors
- Preface
- Section I: B12 Biosynthesis and B12 Chaperones
- Chapter One: Biosynthesis of cobamides: Methods for the detection, analysis and production of cobamides and biosynthetic intermediates
- Abstract
- 1: Introduction
- 2: Extraction, isolation and identification of cobamides
- 3: Isolation of biosynthetic intermediates of cobalamin biosynthesis
- 4: Protocols
- 5: Studying cobamide biosynthesis—Conclusions
- References
- Chapter Two: Guardian of cobamide diversity: Probing the role of CobT in lower ligand activation in the biosynthesis of vitamin B12 and other cobamide cofactors
- Abstract
- 1: Introduction
- 2: Finding new CobT homologs
- 3: Cloning, overexpression, and enzyme purification of CobT homologs
- 4: In vitro reconstitution of the phosphoribosyltransferase activity of a CobT homolog
- 5: Assessing the substrate promiscuity of CobT
- 6: Analysis of regioselectivity of lower ligand activation using asymmetric lower ligands 5-OMeBza and 5-OHBza as examples
- 7: A pairwise substrate competition assay to determine substrate preference
- 8: Conclusions
- Acknowledgment
- References
- Chapter Three: Purification and detection of vitamin B12 analogs
- Abstract
- 1: Introduction
- 2: Cobamide production in bacteria
- 3: Extraction and purification of corrinoids from bacterial cultures
- 4: Methods for quantification and detection of corrinoids
- 5: Appendix
- Acknowledgments
- References
- Further reading
- Chapter Four: A method for the efficient adenosylation of corrinoids
- Abstract
- 1: Introduction to ATP:Co(I)rrinoid adenosyltransferases (ACATs)
- 2: Expression and purification of CobA from Salmonella enterica
- 3: Production and purification of EutT from Listeria monocytogenes in E. coli
- 4: Preparation of corrinoid substrates
- 5: Measuring adenosyltransferase activity
- 6: Conclusions
- References
- Chapter Five: A method for the production, purification and liposome reconstitution of cobamide synthase
- Abstract
- 1: Introduction: B12 and the nucleotide loop assembly (NLA) pathway
- 2: Cloning, overexpression and purification of cobamide synthase (CobS)
- 3: Reconstitution of CobS into liposomes
- 4: Proteoliposome quality controls
- 5: Activity assays
- References
- Chapter Six: A method for the isolation of α-ribazole from vitamin B12, and its enzymatic conversion to α-ribazole 5′-phosphate
- Abstract
- 1: Introduction to coenzyme B12 and to the activation of its nucleobase
- 2: Synthesis and purification of α-Ribazole (α-R)
- 3: Expression and purification of CblS from Geobacillus kaustophilus in E. coli
- 4: Synthesis and purification of α-ribazole-5′-phosphate
- References
- Chapter Seven: The human B12 trafficking chaperones: CblA, ATR, CblC and CblD
- Abstract
- 1: Introduction
- 2: Assays for chaperones in the cytoplasmic branch
- 3: Assays for CblC
- 4: Assays for B12 chaperones in the mitochondrial branch
- Acknowledgment
- References
- Chapter Eight: Antivitamins B12: Synthesis and application as inhibitory ligand of the B12-tailoring enzyme CblC
- Abstract
- 1: Introduction
- 2: Structure-based concept of antivitamins B12
- 3: Synthesis of antivitamins B12 from vitamin B12
- 4: Study of antivitamins B12 as inhibitors of the B12 tailoring enzyme CblC
- 5: Methods
- 6: Biochemical experiments with antivitamins B12 and human CblC
- 7: Crystallographic structure determination of a CblC•F2PhEtyCbl/GSH ternary complex
- 8: Summary
- Acknowledgments
- References
- Section II: B12-dependent enzyme reactions
- Chapter Nine: Coenzyme B12-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase
- Abstract
- 1: Introduction
- 2: Discovery and metabolic roles
- 3: Gene cloning and high-level expression
- 4: Purification and protein-chemical properties
- 5: Enzyme assays and catalytic properties
- 6: Mechanistic studies
- 7: X-ray structure analysis
- 8: Functional analysis of amino acid residues
- 9: Protocol
- 10: Conclusion
- Acknowledgments
- References
- Chapter Ten: Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase
- Abstract
- 1: Introduction
- 2: Mechanism-based inactivation by natural substrates and in situ reactivation
- 3: Discovery and identification of the gene(s) encoding reactivating factors
- 4: Purification, assays, and protein-chemical properties of recombinant reactivating factors
- 5: Mechanistic studies
- 6: X-ray structures of DD-R
- 7: Molecular mechanism of action of reactivating chaperones
- 8: Role of reactivating chaperones in coenzyme recycling: Activity-maintaining system
- 9: Protocol
- 10: Conclusion
- Acknowledgments
- References
- Chapter Eleven: Glutamate mutase and 2-methyleneglutarate mutase
- Abstract
- 1: Introduction
- 2: Roles of glutamate and 2-methyleneglutarate mutases in metabolism
- 3: Properties of glutamate mutase
- 4: Properties of 2-methyleneglutarate mutase
- 5: Crystal structure of glutamate mutase
- 6: EPR spectroscopy
- 7: On the mechanism of glutamate and 2-methyleneglutarate mutases
- 8: Preparation of active glutamate mutase and 2-methyleneglutarate mutase
- 9: Key resources table (ERC)
- 10: Expression of glmS, glmE and mgm without tag in E. coli DH5α
- References
- Chapter Twelve: Human B12-dependent enzymes: Methionine synthase and Methylmalonyl-CoA mutase
- Abstract
- 1: Introduction
- 2: Expression and purification of MCM
- Acknowledgment
- References
- Chapter Thirteen: Heterologous production and biophysical characterization of catabolic Nitratireductor pacificus pht-3B reductive dehalogenase
- Abstract
- 1: Introduction
- 2: N. pacificus pht-3B (NpRdhA) cloning and heterologous expression hosts
- 3: Anaerobic purification and characterization of RdhA reductive dehalogenase
- 4: X-ray crystallography of RdhA proteins
- 5: In vitro NpRdhA enzyme catalysis
- 6: EPR spectroscopy as a tool to study RdhA
- 7: Summary
- References
- Chapter Fourteen: An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH
- Abstract
- 1: Introduction
- 2: Background
- 3: Protocols
- 4: Future directions and opportunities
- 5: Summary
- Acknowledgment
- References
- Edition: 1
- Volume: 668
- Published: May 17, 2022
- Imprint: Academic Press
- No. of pages: 390
- Language: English
- Hardback ISBN: 9780323915915
- eBook ISBN: 9780323915922
NM
Neil Marsh
I am currently Professor of Chemistry and Biological Chemistry at the University of Michigan in Ann Arbor. My research interests center on enzyme mechanisms and protein structure and design. We are currently working on a variety of research projects. In particular, we have a long-standing interest in enzymes that use free radicals to catalyze a variety of unusual chemical transformations. More recently, we have become interested in enzymes involved in hydrocarbon biosynthesis, many of which have novel mechanisms and are of practical interest for the biosynthesis of next-generation biofuels. We are also interested in understanding in molecular detail how enzymes interact with abiological surfaces as this is key to many industrial and biomedical applications where enzymes are immobilized on solid supports. More information about my research can be found here:http://www.lsa.umich.edu/chem/people/faculty/ci.marsheneilg_ci.detail
Affiliations and expertise
Dow Distinguished Faculty Fellow in Sustainability, Professor of Chemistry and Biological Chemistry, Department of Chemistry, University of Michigan, USARead Coenzyme B12 Enzymes Part A on ScienceDirect