AcpM, the Meromycolate Extension Acyl Carrier Protein of Mycobacterium tuberculosis, Is Activated by the 4′- Phosphopantetheinyl Transferase PptT, a Potential Target of the Multistep Mycolic Acid Biosynthesis Oren Zimhony,* ,†,∥ Alon Schwarz, ‡,#,∥ Maria Raitses-Gurevich, ‡ Yoav Peleg, ⊥ Orly Dym, ⊥ Shira Albeck, ⊥ Yigal Burstein, § and Zippora Shakked ‡ † Kaplan Medical Center, Affiliated to the School of Medicine, Hebrew University of Jerusalem and Hadassah Medical Center, POB1 Rehovot 76100, Israel ‡ Department of Structural Biology, ⊥ Israel Structural Proteomics Center, and § Department of Organic Chemistry, Weizmann Institute of Science, 234 Herzl Street, Rehovot 7610001, Israel * S Supporting Information ABSTRACT: 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). Mycobacterium tuberculosis (Mtb) possesses two essential PPTases: acyl carrier protein synthase (Mtb AcpS), which activates the multidomain fatty acid synthase I (FAS I), and Mtb PptT, an Sfp-type broad spectrum PPTase that activates PKSes. To date, it has not been determined which of the two Mtb PPTases, AcpS or PptT, activates the meromycolate extension ACP, Mtb 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 Mtb apo-AcpM to Mtb holo-AcpM by either Mtb PptT or Mtb AcpS. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that Mtb PptT is the PPTase that activates Mtb AcpM. We measured the catalytic activity of Mtb PptT toward CoA, using an activation assay of a blue pigment synthase, BpsA (a nonribosomal peptide synthase, NRPS). BpsA activation by Mtb PptT was inhibited by Mtb apo-AcpM through competition for CoA, in accord with Mtb AcpM activation. A structural model of the putative interaction between Mtb PptT and Mtb AcpM suggests that both hydrophobic and electrostatic interactions stabilize this complex. To conclude, activation of Mtb AcpM by Mtb PptT reveals a potential target of the multistep mycolic acid biosynthesis. M ultidrug-resistant tuberculosis arises as a result of an inappropriate usage and interruption of the relatively long and complex drug regimen required for lasting cure. The therapeutic options for multidrug resistant strains of Mycobacterium tuberculosis (Mtb) are scarce. 1 Thus, there is a need for target identification followed by new drug develop- ment to simplify and shorten therapy, and to prevent the emergence of resistance while providing additional options for treating drug resistant tuberculosis. Mtb possesses a unique lipid-rich cell wall that confers virulence, persistence, and drug resistance, which is comprised mainly of α-alkyl-β-hydroxyl C80-90 fatty acids termed mycolic acids, and several polyketides. 2−5 Several steps in mycolic acid biosynthesis were identified as drug targets for first-line antituberculosis agents. 6 Mycobacteria uniquely between bacteria possess two types of fatty acid synthase systems, FAS I and FAS II and 18 polyeketide synthases (PKSes). 5,6 FAS I is a multifunctional, multidomain protein that elongates acyl-CoA to C16:0−26:0 fatty acids. 7,8 FAS II elongates C16:0−26:0 to C56:0 meromycolate, which may undergo various modifications, and is finally condensed with C26:0 to mycolic acid by PKS13. 6,9,10 The acyl intermediates produced during these processes are attached covalently to an acyl carrier protein (ACP) domain of FAS I or PKS, or to a discrete meromycolate extension ACP of FAS II system, designated Mtb AcpM. 6,11 FAS and PKSes are activated through the transfer of a 4′-phosphopantetheine (4′- PP) group from CoA to an ACP, a reaction that is catalyzed by a4′-PP transferase (PPTase) enzyme. The PPTases have been classified based on sequence and structural features, into three classes. 12,13 The first class includes the ACP synthetases, AcpS-type PPTases, which contain Received: November 21, 2014 Revised: March 6, 2015 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A DOI: 10.1021/bi501444e Biochemistry XXXX, XXX, XXX−XXX