Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2. Evidence for a CobU-GMP intermediate.
The CobU protein of Salmonella typhimurium was overexpressed and purified to approximately 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 approximately 20% 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 (approximately 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.
O'Toole
G A
GA
Department of Bacteriology, University of Wisconsin, Madison 53706, USA.
Escalante-Semerena
J C
JC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003038:Cobamides / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression; D005798:Genes, Bacterial; D006157:Guanosine Monophosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases; D010430:Pentosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D003038:Cobamides; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D006157:Guanosine Monophosphate; D004228:Dithionitrobenzoic Acid; D010430:Pentosyltransferases; C028015:nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase; D009713:Nucleotidyltransferases