Reassessment of the Late Steps of Coenzyme B ₁₂ Synthesis in Salmonella enterica : Evidence that Dephosphorylation of Adenosylcobalamin-5′-Phosphate by the CobC Phosphatase Is the Last Step of the Pathway

Abstract

We report that cobC strains of Salmonella enterica serovar Typhimurium are impaired in the ability to salvage cobyric acid (Cby), a de novo corrin ring biosynthetic intermediate, under aerobic growth conditions. In vivo and in vitro evidence support the conclusion that this new phenotype of cobC strains is due to the inability of serovar Typhimurium to dephosphorylate adenosylcobalamin-5-phosphate (AdoCbl-5-P), the product of the condensation of ␣-ribazole-5-phosphate (␣-RP) and adenosylcobinamide-GDP by the AdoCbl-5-P synthase (CobS, EC 2.7.8.26) enzyme. Increased flux through the 5,6-dimethylbenzimidazole and cobinamide (Cbi) activation branches of the nucleotide loop assembly pathway in cobC strains restored AdoCbl-5-P synthesis from Cby in a cobC strain. The rate of the CobS-catalyzed reaction was at least 2 orders of magnitude higher with ␣-RP than with ␣-ribazole as substrate. On the basis of the data reported herein, we conclude that removal of the phosphoryl group from AdoCbl-5-P is the last step in AdoCbl biosynthesis in serovar Typhimurium and that the reaction is catalyzed by the AdoCbl-5-P phosphatase (CobC) enzyme. Explanations for the correction of the Cby salvaging phenotype are discussed.

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

“…A remarkable example is the presence of a complete biosynthetic pathway for cobalamin, an essential cofactor for OHR metabolism. Indeed, based on the anaerobic pathway described by Roessner & Scott [59] and the cobinamide salvaging pathway studied by EscalanteSemerena and co-workers [43,[60][61][62], all genes were clearly identified in D. restrictus, although it cannot grow without a supply of vitamin B 12 in the medium ( [1]; J. Maillard 2012, unpublished data). The proteomic data obtained here showed that about half of the proteins of the biosynthetic pathway mostly from the upper pathway were not detected, indicating that under the growth conditions applied, D. restrictus used the corrinoid amended and possibly modified according to its needs.…”

Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

“…Related to this, TunG, a putative phosphoglycerate mutase with homology to Francci3_2350, is potentially a CobC-type alpha-ribazole-5′-phosphate phosphatase. CobC is involved in the biosynthesis of cobalamin (vitamin B12), wherein the dephosphorylation of adenosylcobalamin-5′-phosphate by the CobC phosphatase is the last step of the pathway (Zayas and Escalante-Semerena, 2007). Since cobalamin is a co-factor for the radical SAM proteins (Tun B), it is quite possible that the TunG promotes the production of cobalamin for its use during the biosynthesis of tumicamycin.…”

Biosynthesis of Vitamins by Probiotic Bacteria

“…CobZ was also found to have a flavin in the form of a non-covalently bound FAD, two Fe-S centers, and a b-type heme, which was not similar to CobG [51]. It was thought that the final step in the cobalamin biosynthetic pathway in S. typhimurium involved the dephosphorylation of adenosylcobalamin-5′-phosphate, which is catalyzed by CobC and challenges the pathway indicated where CobS catalyzes the condensation of a-ribazole and an Ado-GDP-cobinamide [52]. The gene that reduces cobalt in the aerobic pathway has yet to be identified, but two candidate genes were identified to encode this enzyme, named CobR [53].…”