Molecular characterization of the 4′-phosphopantothenoylcysteine synthetase domain of bacterial Dfp flavoproteins

In bacteria, coenzyme A is synthesized in five steps from pantothenate. The flavoprotein Dfp catalyzes the synthesis of the coenzyme A precursor 4'-phosphopantetheine in the presence of 4'-phosphopantothenate, cysteine, CTP, and Mg2+ (Strauss, E., Kinsland, C., Ge, Y., McLafferty, F. W., and Begley, T. P. (2001) J. Biol. Chem. 276,13513-13516). It has been shown that the NH2-terminal domain of Dfp has 4'-phosphopantothenoylcysteine decarboxylase activity (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838-31846). Here I demonstrate that the COOH-terminal CoaB domain of Dfp catalyzes the synthesis of 4'-phosphopantothenoylcysteine. The exchange of conserved amino acid residues within the CoaB domain revealed that the synthesis of 4'-phosphopantothenoylcysteine occurs in two half-reactions. Using the mutant protein His-CoaB N210D the putative acyl-cytidylate intermediate of 4'-phosphopantothenate was detectable. The same intermediate was detectable for the wild-type CoaB enzyme if cysteine was omitted in the reaction mixture. Exchange of the conserved Lys(289) residue, which is part of the strictly conserved (KXKK292)-K-289 motif of the CoaB domain, resulted in complete loss of activity with neither the acyl-cytidylate intermediate nor 4'-phosphopantothenoylcysteine being detectable. Gel filtration experiments indicated that CoaB forms dimers. Residues that are important for dimerization are conserved in CoaB proteins from eubacteria, Archaea, and eukaryotes.