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 Mg(2+) (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 NH(2)-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 (289)KXKK(292) 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.
Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany. Thomas.Kupke@t-online.de
Thomas
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Kupke
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D006639:Histidine; D010453:Peptide Synthases; C022374:phosphopantothenoyl-cysteine synthetase; D008239:Lysine; D003065:Coenzyme A