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 purif ied to similar to 94% homogeneity. N-terminal sequencing of purified C obU confirmed the first 22 amino acids, In vitro assays showed that Co bU has kinase and guanylyltransferase activities which catalyze the sy nthesis of adenosyl-cobinamide-GDP from adenosyl-cobinamide, via an ad enosyl-cobinamide-phosphate intermediate. We present evidence that the transfer of the guanylyl moiety of GTP to adenosyl-cobinamide-phospha te proceeds via an phosphoramidate linked, enzyme guanylyl intermediat e, In the presence of oxygen, kinase and guanylyltransferase activitie s of CobU were lost. Treatment of inactive CobU with dithiothreitol re stored 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) a nd N-ethylmaleimide abolished both CobU activities, Native CobU protei n 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 activ ities were determined, The preferred corrinoid substrate for this enzy me was adenosyl-cobinamide. In vitro experiments are consistent with p revious genetic studies which had suggested that adenosyl-cobinamide w as the preferred substrate of CobU, and that CobU functioned more effi ciently in the absence of oxygen.