Purification and Characterization of the Bifunctional CobU Enzyme of Salmonella typhimurium LT2

Abstract

The CobU protein of Salmonella typhimurium was overexpressed and purified to ϳ94% homogeneity. Nterminal sequencing of purified CobU confirmed the first 22 amino acids. In vitro assays showed that CobU has kinase and guanylyltransferase activities which catalyze the synthesis of adenosyl-cobinamide-GDP from adenosyl-cobinamide, via an adenosyl-cobinamidephosphate intermediate. We present evidence that the transfer of the guanylyl moiety of GTP to adenosylcobinamide-phosphate proceeds via an phosphoramidate-linked, enzyme-guanylyl intermediate.In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost. Treatment of inactive CobU with dithiothreitol restored ϳ20% of the kinase and guanylyltransferase activities, indicating the involvement of sulfhydryl groups in enzyme activity. The sulfhydryl modifying agents 5,5-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide abolished both CobU activities.Native CobU protein was a dimer (ϳ40 kDa) that functioned optimally at pH 8.8 -9.0 and 37°C. Substrates and kinetic parameters for both activities were determined. The preferred corrinoid substrate for this enzyme was adenosyl-cobinamide. In vitro experiments are consistent with previous genetic studies which had suggested that adenosyl-cobinamide was the preferred substrate of CobU, and that CobU functioned more efficiently in the absence of oxygen.

Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon

“…The homology of PrpB to CPEP mutase of S. hygroscopicus is most intriguing given the fact that CPEP mutase catalyzes the synthesis of a phosphonate (C-P) bond (13,23). A phosphonate intermediate has not been proposed in any of the known pathways for propionate breakdown (7,38), and we feel it unlikely that the propionate catabolic pathway in S. typhimurium involves a phosphonate compound.…”

“…The resulting E. coli strains were maintained at 30ЊC in Luria-Bertani broth containing ampicillin and kanamycin. Overexpression protocols have been described elsewhere (23).…”

The last step in coenzyme B12 synthesis is localized to the cell membrane in bacteria and archaea

“…A cell-free extract was prepared as previously described from cells from a culture of E. coli BL21(DE3) carrying plasmid pCOBS4 harvested 4 h after induction (O'Toole & Escalante-Semerena, 1995). Purification of the overproduced protein was monitored by SDS-PAGE (Laemmli, 1970).…”

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

“…Protocols for the synthesis and purification of AdoCbi-GDP and ␣-RP have been described (35,37). In this work, we used an Alltima HP C 18 AQ 5 column (150 by 4.6 mm) (Alltech) and a Waters 600 HPLC system to resolve products from reagents.…”