title: 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.
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.
authors.affiliation: †Kaplan Medical Center, Affiliated to the School of Medicine, Hebrew University of Jerusalem and Hadassah Medical Center, POB1 Rehovot 76100, Israel.
authors.forename: Oren
authors.initials: O
authors.lastname: Zimhony
authors.forename: Alon
authors.initials: A
authors.lastname: Schwarz
authors.forename: Maria
authors.initials: M
authors.lastname: Raitses-Gurevich
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peleg
authors.forename: Orly
authors.initials: O
authors.lastname: Dym
authors.forename: Shira
authors.initials: S
authors.lastname: Albeck
authors.forename: Yigal
authors.initials: Y
authors.lastname: Burstein
authors.forename: Zippora
authors.initials: Z
authors.lastname: Shakked
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D003065:Coenzyme A / Q000737:chemistry; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009171:Mycolic Acids / Q000378:metabolism*; D010453:Peptide Synthases / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry*
chemical_list: C431823:AcpM protein, Mycobacterium tuberculosis; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009171:Mycolic Acids; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D003065:Coenzyme A