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An Aminoimidazole Radical Intermediate in the Anaerobic Biosynthesis of the 5,6-Dimethylbenzimidazole Ligand to Vitamin B12.

ABSTRACT: Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathways depending on the presence of oxygen in the environment. The anaerobic biosynthesis pathway of 5,6-dimethylbenzimidazole, the so-called "lower ligand" to the cobalt center, has been recently identified. This process begins with the conversion of 5-aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (HBI) by the radical S-adenosyl-l-methionine (SAM) enzyme BzaF, also known as HBI synthase. In this work we report the characterization of a radical intermediate in the reaction of BzaF using electron paramagnetic resonance spectroscopy. Using various isotopologues of AIR, we extracted hyperfine parameters for a number of nuclei, allowing us to propose plausible chemical compositions and structures for this intermediate. Specifically, we find that an aminoimidazole radical is formed in close proximity to a fragment of the ribose ring. These findings induce the revision of past proposed mechanisms and illustrate the ability of radical SAM enzymes to tightly control the radical chemistry that they engender.

SUBMITTER: Gagnon DM

PROVIDER: S-EPMC6435355 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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An Aminoimidazole Radical Intermediate in the Anaerobic Biosynthesis of the 5,6-Dimethylbenzimidazole Ligand to Vitamin B12.

Gagnon Derek M DM Stich Troy A TA Mehta Angad P AP Abdelwahed Sameh H SH Begley Tadhg P TP Britt R David RD

Journal of the American Chemical Society 20180927 40

Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathways depending on the presence of oxygen in the environment. The anaerobic biosynthesis pathway of 5,6-dimethylbenzimidazole, the so-called "lower ligand" to the cobalt center, has been recently identified. This process begins with the conversion of 5-aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (HBI) by the radical S-adenosyl-l-methionine (SAM) enzyme BzaF, also known as HBI synthase. In t ...[more]

PMID: 30208703

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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

An Aminoimidazole Radical Intermediate in the Anaerobic Biosynthesis of the 5,6-Dimethylbenzimidazole Ligand to Vitamin B12.

Publications

An Aminoimidazole Radical Intermediate in the Anaerobic Biosynthesis of the 5,6-Dimethylbenzimidazole Ligand to Vitamin B12.

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