PURIFICATION AND CHARACTERIZATION OF THE BIFUNCTIONAL COBU ENZYME OF SALMONELLA-TYPHIMURIUM LT2 - EVIDENCE FOR A COBU-SIMILAR-TO-GMP INTERMEDIATE

The CobU protein of Salmonella typhimurium was overexpressed and purified to similar to 94% homogeneity. N-terminal 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-cobinamide-phosphate intermediate. We present evidence that the transfer of the guanylyl moiety of GTP to adenosyl-cobinamide-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 similar to 20% of the kinase and guanylyltransferase activities, indicating the involvement of sulfhydryl groups in enzyme activity. The sulfhydryl modifying agents 5,5'-dithiobis(2-nitro-benzoic acid) and N-ethylmaleimide abolished both CobU activities, Native CobU protein was a dimer (similar to 40 kDa) that functioned optimally at pH 8.8-9.0 and 37 degrees 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.