Fluorinated vitamin B12 analogs are cofactors of corrinoid-dependent enzymes: A 19F-labeled nuclear magnetic resonance probe for identifying corrinoid-protein interactions

Abstract

The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B12 analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4- fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B12. Hence, it is possible to use 19F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.