cobamamide and cobinamide

The ability of archaea to salvage cobinamide has been under question because archaeal genomes lack orthologs to the bacterial nucleoside triphosphate:5'-deoxycobinamide kinase enzyme (cobU in Salmonella enterica). The latter activity is required for cobinamide salvaging in bacteria. This paper reports evidence that archaea salvage cobinamide from the environment by using a pathway different from the one used by bacteria. These studies demanded the functional characterization of two genes whose putative function had been annotated based solely on their homology to the bacterial genes encoding adenosylcobyric acid and adenosylcobinamide-phosphate synthases (cbiP and cbiB, respectively) of S. enterica. A cbiP mutant strain of the archaeon Halobacterium sp. strain NRC-1 was auxotrophic for adenosylcobyric acid, a known intermediate of the de novo cobamide biosynthesis pathway, but efficiently salvaged cobinamide from the environment, suggesting the existence of a salvaging pathway in this archaeon. A cbiB mutant strain of Halobacterium was auxotrophic for adenosylcobinamide-GDP, a known de novo intermediate, and did not salvage cobinamide. The results of the nutritional analyses of the cbiP and cbiB mutants suggested that the entry point for cobinamide salvaging is adenosylcobyric acid. The data are consistent with a salvaging pathway for cobinamide in which an amidohydrolase enzyme cleaves off the aminopropanol moiety of adenosylcobinamide to yield adenosylcobyric acid, which is converted by the adenosylcobinamide-phosphate synthase enzyme to adenosylcobinamide-phosphate, a known intermediate of the de novo biosynthetic pathway. The existence of an adenosylcobinamide amidohydrolase enzyme would explain the lack of an adenosylcobinamide kinase in archaea.

Topics: Amidohydrolases; Archaeal Proteins; Cobamides; Genes, Archaeal; Genetic Complementation Test; Halobacterium

The recent structures of cobalamin-dependent methionine synthase and methylmalonyl-CoA mutase have revealed a striking conformational change that accompanies cofactor binding to these proteins. Alkylcobalamins have octahedral geometry in solution at physiological pH, and the lower axial coordination position is occupied by the nucleotide, dimethylbenzimidazole ribose phosphate, that is attached to one of the pyrrole rings of the corrin macrocycle via an aminopropanol moiety. In contrast, in the active sites of these two B12-dependent enzymes, the nucleotide tail is held in an extended conformation in which the base is far removed from the cobalt in cobalamin. Instead, a histidine residue donated by the protein replaces the displaced intramolecular base. This unexpected mode of cofactor binding in a subgroup of B12-dependent enzymes has raised the question of what role the nucleotide loop plays in cofactor binding and catalysis. To address this question, we have synthesized and characterized two truncated cofactor analogues: adenosylcobinamide and adenosylcobinamide phosphate methyl ester, lacking the nucleotide and nucleoside moieties, respectively. Our studies reveal that the nucleotide tail has a modest effect on the strength of cofactor binding, contributing approximately 1 kcal/mol to binding. In contrast, the nucleotide has a profound influence on organizing the active site for catalysis, as evidenced by the retention of the base-off conformation in the truncated cofactor analogues bound to the mutase and by their inability to support catalysis. Characterization of the kinetics of adenosylcobalamin (AdoCbl) binding by stopped-flow fluorescence spectroscopy reveals a pH-sensitive step that titrates to a pKa of 7.32 +/- 0.19 that is significantly different from the pKa of 3.7 for dimethylbenzimidazole in free AdoCbl. In contrast, the truncated cofactors associate very rapidly with the enzyme at rates that are too fast to measure. Based on these observations, we propose a model in which the base-on to base-off conformational change is slow and is assisted by the enzyme, and is followed by a rapid docking of the cofactor in the active site.

Topics: Benzimidazoles; Binding Sites; Catalysis; Cobamides; Kinetics; Methylmalonyl-CoA Mutase; Propionibacterium; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Tryptophan

Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was estimated to be 8.6 X 10(9) and 9.6 X 10(6) L/mol, respectively. The association constant for adenosylcobalamin, methylcobalamin, and hydroxocobalamin was indirectly determined, and values of 2.5 X 10(5), 1.7 X 10(9), and 2.3 X 10(9) L/mol, respectively, were obtained. Photolysis in the presence of potassium cyanide rendered each of the three cobalamins equivalent to cyanocobalamin in immunoreactivity. The mean concentration of cobalamin in normal human sera and cobalamin-deficient sera measured as cyanocobalamin by radioimmunoassay using this anticobalamin antibody was significantly lower than the concentration measured in the same extracts by competitive ligand-binding radioassays using intrinsic factor and transcobalamin I. These findings, although indirect, support the proposition that there may be factor(s) in normal and cobalamin-deficient sera that falsely elevate the concentration of true cobalamin if the radioassay uses R protein as the binder. However, the lower concentration of serum cobalamin measured by radioimmunoassay compared with the intrinsic factor radioassay also indicates that this "purported" factor(s) reacts to some extent with intrinsic factor but not with the cobalamin antibody.

Topics: Antibody Formation; Antibody Specificity; Cobamides; Cross Reactions; Humans; Hydroxocobalamin; Mathematics; Radioimmunoassay; Radioligand Assay; Reagent Kits, Diagnostic; Vitamin B 12