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In bacteria, coenzyme A is synthesized in five steps from D-pantothenate. The Dfp flavoprotein catalyzes the synthe-sis of the coenzyme A precursor 4¢-phosphopantetheine from 4¢-phosphopantothenate and cysteine using the cofac-tors CTP and flavine mononucleotide via the phospho-peptide-like compound 4¢-phosphopantothenoylcysteine. The synthesis of 4¢-phosphopantothenoylcysteine is cata-lyzedby theC-terminal CoaBdomainofDfp andoccurs via the acyl-cytidylate intermediate 4¢-phosphopantothenoyl-CMP in two half reactions. . | Eur. J. Biochem. 271 163-172 2004 FEBS 2003 doi 10.1046 j.1432-1033.2003.03916.x Active-site residues and amino acid specificity of the bacterial 4 -phosphopantothenoylcysteine synthetase CoaB Thomas Kupke Lehrstuhl fur Mikrobielle Genetik Universitat Tubingen Tubingen Germany In bacteria coenzyme A is synthesized in five steps from D-pantothenate. The Dfp flavoprotein catalyzes the synthesis of the coenzyme A precursor 4 -phosphopantetheine from 4 -phosphopantothenate and cysteine using the cofactors CTP and flavine mononucleotide via the phosphopeptide-like compound 4 -phosphopantothenoylcysteine. The synthesis of 4 -phosphopantothenoylcysteine is catalyzed by the C-terminal CoaB domain of Dfp and occurs via the acyl-cytidylate intermediate 4 -phosphopantothenoyl-CMP in two half reactions. In this new study the molecular characterization of the CoaB domain is continued. In addition to the recently described residue Asn210 two more active-site residues Arg206 and Ala276 were identified and shown to be involved in the second half reaction of the R -4 -phospho-N-pantothenoylcysteine synthetase. The proposed intermediate of the R -4 -phospho-N-panto-thenoylcysteine synthetase reaction 4 -phosphopantothe- noyl-CMP was characterized by MALDI-TOF MS and it was shown that the intermediate is copurified with the mutant His-CoaB N210H K proteins. Therefore His-CoaB N210H and His-CoaB N210K will be of interest to elucidate the crystal structure of CoaB complexed with the reaction intermediate. Wild-type His-CoaB is not absolutely specific for cysteine and can couple derivatives of cysteine to 4 -phosphopantothenate. However no phosphopeptide-like structure is formed with serine. Molecular characterization of the temperature-sensitive Escherichia coli dfp-1 mutant revealed that the residue adjacent to Ala276 Ala275 of the strictly conserved AAVAD 275-279 motif is exchanged for Thr. Keywords coenzyme A biosynthesis 4 -phosphopantethe-ine 4 -phosphopantothenoylcysteine .