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Coenzyme B12 enzymes. Part A / edited by E. Neil G. Marsh. -- Cambridge, MA : Academic Press, an imprint of Elsevier, 2022. – (58.17435/C719/v.668) |
Contents
Contributors
Preface
Section I
B12 Biosynthesis and B12 Chaperones
1. Biosynthesis of cobamides: Methods for the detection, analysis and production of cobamides and biosynthetic intermediates
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
2. Guardian of cobamide diversity: Probing the role of CobT in lower ligand activation in the biosynthesis of vitamin B12 and other cobamide cofactors
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
3. Purification and detection of vitamin B12 analogs
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
4. A method for the efficient adenosylation of corrinoids
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
Acknowledgments
References
5. A method for the production, purification and liposome reconstitution of cobamide synthase
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
6. A method for the isolation of α-ribazole from vitamin B12, and its enzymatic conversion to a-ribazole 5'-phosphate
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
7. The human B12 trafficking chaperones: CblA, ATR, CblC and CblD
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
8. Antivitamins B12: Synthesis and application as inhibitory ligand of the B12-tailoring enzyme CblC
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
9. Coenzyme B12-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase
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
10. Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase
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
11. Glutamate mutase and 2-methyleneglutarate mutase
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. coil DH5α
References
12. Human B12-dependent enzymes: Methionine synthase and Methylmalonyl-CoA mutase
1. Introduction
2. Expression and purification of MCM
Acknowledgment
References
13. Heterologous production and biophysical characterization of catabolic Nitratireductor pacificus pht-3B reductive dehalogenase
1. Introduction
2. IV. 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
14. An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH
1. Introduction
2. Background
3. Protocols
4. Future directions and opportunities
5. Summary
Acknowledgment
References
