Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium.
The cobA gene of Salmonella typhimurium and its product were overexpressed to approximately 20% of the total cell protein. CobA was purified to 98% homogeneity; N-terminal sequence analysis (21 residues) of homogeneous protein confirmed the predicted amino acid sequence. ATP:corrinoid adenosyltransferase activity was demonstrated in vitro to be associated with CobA. This activity was optimal at pH 8 and 37 degrees C. A quantitative preference was determined for Mn(II) cations and ATP. The apparent Km of CobA for ATP was 2.8 microM, and that for cob(I)alamin was 5.2 microM. Vmax was measured at 0.43 nmol/min. Cobinamide served as the substrate for CobA to yield adenosylcobinamide. Activity was stable at 4 degrees C for several weeks but was lost rapidly at room temperature (50% overnight). Dithiothreitol was required to maintain the enzymatic activity of CobA.
Department of Bacteriology, University of Wisconsin-Madison 53706-1567.
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D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002413:Cations, Divalent; D003038:Cobamides / Q000096:biosynthesis*; D005798:Genes, Bacterial / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D017421:Sequence Analysis; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003038:Cobamides; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C082269:ATP-corrinoid adenosyltransferase; C000913:cobamamide