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-pantothenoylcysteine synthetase reaction, 4'-phosphopantothenoyl-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.