AcpM, the Meromycolate Extension Acyl Carrier Protein of <i>Mycobacterium tuberculosis</i>, Is Activated by the 4′-Phosphopantetheinyl Transferase PptT, a Potential Target of the Multistep Mycolic Acid Biosynthesis

Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4′-phosphopantetheine (4′-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4′-PP is mediated by 4′ phosphopantetheinyl transfersases (PPTases). <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) possesses two essential PPTases: acyl carrier protein synthase (<i>Mtb</i> AcpS), which activates the multidomain fatty acid synthase I (FAS I), and <i>Mtb</i> PptT, an Sfp-type broad spectrum PPTase that activates PKSes. To date, it has not been determined which of the two <i>Mtb</i> PPTases, AcpS or PptT, activates the meromycolate extension ACP, <i>Mtb</i> AcpM, en route to the production of mycolic acids, the main components of the mycobacterial cell wall. In this study, we tested the enzymatic activation of a highly purified <i>Mtb</i> apo-AcpM to <i>Mtb</i> holo-AcpM by either <i>Mtb</i> PptT or <i>Mtb</i> AcpS. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that <i>Mtb</i> PptT is the PPTase that activates <i>Mtb</i> AcpM. We measured the catalytic activity of <i>Mtb</i> PptT toward CoA, using an activation assay of a blue pigment synthase, BpsA (a nonribosomal peptide synthase, NRPS). BpsA activation by <i>Mtb</i> PptT was inhibited by <i>Mtb</i> apo<i>-</i>AcpM through competition for CoA, in accord with <i>Mtb</i> AcpM activation. A structural model of the putative interaction between <i>Mtb</i> PptT and <i>Mtb</i> AcpM suggests that both hydrophobic and electrostatic interactions stabilize this complex. To conclude, activation of <i>Mtb</i> AcpM by <i>Mtb</i> PptT reveals a potential target of the multistep mycolic acid biosynthesis