Pseudocoenzyme B12 and Adenosyl-Factor A: Electrochemical Synthesis and Spectroscopic Analysis of Two Natural B12 Coenzymes with Predominantly ‘Base-off' Constitution

Fieber, Wolfgang; Hoffmann, Bernd; Schmidt, Wolfgang; Stupperich, Erhard; Konrat, Robert; Kräutler, Bernhard

doi:10.1002/1522-2675(200203)85:3<927::aid-hlca927>3.0.co;2-a

Cited by 32 publications

(34 citation statements)

References 17 publications

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“…In aqueous solution, 4 and 5 exist mainly in the base-off form, as deduced from UV/Vis, CD, and 1 H NMR spectroscopy. [20,21] As expected, these baseoff adenosylcobamides bind 3 to 7 times weaker than 1. Clearly, the constitution of the nucleotide base and its intramolecular interaction with the cobalt center play important roles in the recognition of corrinoids by antibody 2C2.…”

supporting

confidence: 58%

“…Vitamin B 12 derivatives show diagnostic absorbances at wavelengths longer than 300 nm, [21,22] which are easily monitored in the presence of the colorless antibody. Addition of 2C2 to coenzyme B 12 (1) caused a small shift in the visible part of the UV/Vis spectrum (from 524 to 536 nm) with an 8 % decrease of the extinction coefficient.…”

mentioning

confidence: 99%

“…These results suggest that the complete cobalt-coordinating nucleotide loop is required for formation of the B 12 ±antibody complex. To test this hypothesis, pseudocoenzyme B 12 (4) [20,21] and Co b -5'-deoxyadenosyl factor A (5) [21,22] were also investigated as competitive inhibitors. These natural coenzyme B 12 analogues differ from 1 by the replacement of the DMB base by adenine and 2-methyladenine, respectively (Scheme 1).…”

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confidence: 99%

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An Antibody that Reconstitutes the “Base-On” Form of B12 Coenzymes

Hannak

Konrat

Schuler

et al. 2002

Angew. Chem. Int. Ed.

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The cobalt-coordinating 5,6-dimethylbenzimidazole (DMB) nucleotide is a structural feature of coenzyme B 12 and of methylcobalamin [1,2] that controls the reactivity of these organometallic cofactors. [3,4] The DMB nucleotide and the functionalized corrin ring are both important for tight and selective binding to B 12 apoenzymes. Several coenzyme B 12 dependent enzymes were recently shown to bind the B 12 cofactor in an unanticipated ™base-off/His-on∫ form, [5±7] in which the DMB nucleotide is displaced from the cobalt center by a histidine residue from the protein. In contrast, diol dehydratase binds coenzyme B 12 ™base-on∫, as described in a crystal structure at the end of the 1990s. [8] Here we report on a monoclonal antibody raised against coenzyme B 12 , which binds the ™base-on∫ form of natural coenzyme B 12 analogues that prefer to be ™base-off∫ in solution. This B 12 antibody thus causes an unprecedented ™reverse∫ coordinative ™base-off∫ to ™base-on∫ reconstitution, which also results in a significant change in the reactivity of the bound B 12 coenzyme.Over the last 15 years, antibodies have proved useful as catalysts for a set of remarkable chemical reactions and as receptors for probing ligand-binding mechanisms. [9±14] Versatile cofactors such as coenzyme B 12 have considerable potential to extend the chemistry of these systems. To investigate the ability of antibodies to recognize and modulate the chemical reactivity of corrinoid coenzymes, we have raised monoclonal antibodies against coenzyme B 12 (1). Although other antibodies that recognize B 12 derivatives are known, [15] their binding properties were never scrutinized spectroscopically.For immunization, coenzyme B 12 (1) was coupled to the carrier protein thyroglobulin (TG) with a succinic acid linker [16] (48 haptens/TG). An immune response was elicited against the TG±hapten conjugate, and a panel of monoclonal antibodies with good antigen recognition was prepared and purified by conventional means. [17] After initially screening 20 antigen binders by competition ELISA (enzyme-linked immunosorbent assay), [18] antibody 2C2 was selected for detailed characterization because it recognized both the DMB nucleotide and the corrin ring.Antibody 2C2 binds 1 with an estimated dissociation constant (K d ) of 9.8 AE 2.6 mm. As summarized in Table 1, vitamin B 12 (2; Scheme 1) and several B 12 derivatives compete effectively with the antigen. Dicyanocobinamide (3), an analogue of 1 and 2 lacking the nucleotide function, is the weakest corrinoid ligand tested, and neither benzimidazole nor 2-amino-isopropylribazole phosphate [19] alone compete with the antigen. These results suggest that the complete cobalt-coordinating nucleotide loop is required for formation of the B 12 ±antibody complex. To test this hypothesis, pseudocoenzyme B 12 (4) [20,21] and Co b -5'-deoxyadenosyl factor A (5) [21,22] were also investigated as competitive inhibitors. These natural coenzyme B 12 analogues differ from 1 by the replacement of the DMB base by adenine and 2-methylade...

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confidence: 99%

mentioning

confidence: 99%

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An Antibody that Reconstitutes the “Base-On” Form of B12 Coenzymes

Hannak

Konrat

Schuler

et al. 2002

Angew. Chem. Int. Ed.

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“…The two latter substances presented an interesting example of trans-axial effect, where the ligand coordinated to one side of the ring affected interactions at the opposite side via the cobalt ion. It is known that the efficient donation of electrons from the upper Ado-group to the Co-ion causes detachment of the nucleotide base from the lower surface of pseudo-B 12 and factor A [2,14]. It seems that the binding of tetrazole in place of the dissociated nucleotide was also precluded.…”

Section: Interaction Of Different Corrinoids With Stzmentioning

confidence: 99%

Tetrazole derivatives and matrices as novel cobalamin coordinating compounds

Fedosov

Berglund

Nexø

et al. 2007

Journal of Organometallic Chemistry

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Cobalamin (Cbl, vitamin B 12 ) consists of two moieties: (i) the corrin ring with the central Co-ion in the oxidation states Co 3+/2+/1+ and (ii) the nucleotide side chain. The lower position of the ring is typically occupied by the nucleotide base (Bzm), whereas the upper surface coordinates exchangeable ligands. We have found that amino-tetrazole can coordinate to H 2 O AE Cbl (Co 3+ ) with K d = 10 À5 -10 À6 M. A specific group (presumably tetrazole, TZ) can be easily created in CNBr-activated Sepharose by treatment with N À 3 . The prepared matrix (STZ) contained %10 mM of the active groups, which bound H 2 O AE corrinoids with K d = 10 À5 -10 À6 M. Stability of STZCbl bonds gradually increased and reached K d = 10 À7 M over 10-20 h (20°C, pH 6-7). This effect can be ascribed to partial displacement of Bzm and coordination of TZ to the lower position. The binding was most efficient at pH 4-7 and low ionic strength, yet, noticeable adsorption took place even at extreme conditions, pH 1-9 and I = 0-2 M. Reduced corrins (Co 2+ ) also exhibited high affinity for STZ. The bound ligands could be eluted as H 2 O AE Cbl (pH 0), HO AE Cbl (pH 14) or diCN AE Cbl (pH 9-12, CN À ). The adsorbent is applicable for one-step purification of corrins from a crude extract; separation of aquo-and diaquo-forms; specific capturing of H 2 O AE Cbl from a mixture containing organo-Cbls or protein-bound Cbl, analysis of peptide-Cbl dissociation kinetics, etc.

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“…Corrinoid Binding Assay-The ability of trout cobalaminbinding protein to bind the corrinoids cyano-cobalamin (Sigma), dicyano-cobinamide (Sigma), and adenosyl-pseudocobalamin (synthesized and described elsewhere (15)) was explored using a competitive assay previously described (16) with corrinoid concentrations ranging from 0 to 355 nM.…”

mentioning

confidence: 93%

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

Greibe

Fedosov

Sørensen

et al. 2012

Journal of Biological Chemistry

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Background: Three cobalamin-binding proteins are expressed in most mammals. The expression in lower vertebrates is poorly explored. Results: We identified a single cobalamin-binding protein in the rainbow trout. The protein behaves as a structural hybrid between the three known mammalian binders. Conclusion: A novel cobalamin-binding protein present in fish has been identified. Significance: This study provides insights into the phylogenetics of cobalamin-binding proteins.

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Pseudocoenzyme B12 and Adenosyl-Factor A: Electrochemical Synthesis and Spectroscopic Analysis of Two Natural B12 Coenzymes with Predominantly ‘Base-off' Constitution

Cited by 32 publications

References 17 publications

An Antibody that Reconstitutes the “Base-On” Form of B12 Coenzymes

An Antibody that Reconstitutes the “Base-On” Form of B12 Coenzymes

Tetrazole derivatives and matrices as novel cobalamin coordinating compounds

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

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