The stepwise truncation of CoA. (A) Representation of the incremental truncation of CoA by ThnR, ThnH, and ThnT. ThnR cleaves CoA to 4-phosphopantetheine, whereas ThnH subsequently cleaves the phosphate to produce pantetheine, and ThnT hydrolyzes pantetheine to yield cysteamine. (B–E) HPLC analyses of coupled enzyme assays to cleave CoA before CMQT derivatization: ThnH and ThnT (B), ThnR and ThnT, ThnR and ThnH (D), and ThnR, ThnH, and ThnT (E).The 4-phosphopantetheine is labeled as 4-P-Pant.; pantetheine is labeled as Pant.; cysteamine is labeled as Cyst. Peaks corresponding to enzymatic products are highlighted in red.