Dataset Information

Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the lower ligand of B12.

ABSTRACT: The synthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of coenzyme B(12), has remained elusive. We report in vitro and in vivo evidence that the BluB protein of the photosynthetic bacterium Rhodospirillum rubrum is necessary and sufficient for catalysis of the O(2)-dependent conversion of FMNH(2) to DMB. The product of the reaction (DMB) was isolated by using reverse-phase high-pressure liquid chromatography, and its identity was established by UV-visible spectroscopy and MS. No metals were detected in homogeneous preparations of BluB, and the enzyme did not affect DMB synthesis from 4,5-dimethylphenylenediamine and ribose-5-phosphate. The effect of the lack of bluB function in R. rubrum was reflected by the impaired ability of a DeltabluB strain to convert Mg-protoporphyrin IX monomethyl ester (MPE) into protochlorophylide, a reaction of the bacteriochlorophyll biosynthetic pathway catalyzed by the MPE-cyclase enzyme present in this bacterium (BchE, EC 1.14.13.81), a predicted coenzyme B(12)-dependent enzyme. The growth defect of the DeltabluB strain observed under anoxic photoheterotrophic conditions was corrected by the addition of DMB or B(12) to the culture medium or by introducing into the strain a plasmid encoding the wild-type allele of bluB. The findings reported here close an important gap in our understanding of the enzymology of the assembly of coenzyme B(12).

SUBMITTER: Gray MJ

PROVIDER: S-EPMC1815282 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the lower ligand of B12.

Gray Michael J MJ Escalante-Semerena Jorge C JC

Proceedings of the National Academy of Sciences of the United States of America 20070214 8

The synthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of coenzyme B(12), has remained elusive. We report in vitro and in vivo evidence that the BluB protein of the photosynthetic bacterium Rhodospirillum rubrum is necessary and sufficient for catalysis of the O(2)-dependent conversion of FMNH(2) to DMB. The product of the reaction (DMB) was isolated by using reverse-phase high-pressure liquid chromatography, and its identity was established by UV-visible spectroscopy and MS. No meta ...[more]

PMID: 17301238

Similar Datasets

Project description:The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12). Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12. In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon. The cobT gene is the last gene in the operon. Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame. Both the CobT protein of S. typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB. This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide. Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype. J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante Semerena have suggested (J. Bacteriol. 176:3568-3575, 1994) that S. typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity. We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate. Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation. Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide. Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype. Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU). Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.

Dataset Information

Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the lower ligand of B12.

Publications

Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the lower ligand of B12.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets