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.

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis

“…Both the Bacillus subtilis and human amino‐adipate semialdehyde dehydrogenase phosphopantetheinyl transferases depend on hydrophobic residues that flank their active sites in order to facilitate the hydrophobic interactions involved in binding their carrier protein substrates . Furthermore, the shape of the active‐site flanking hydrophobic surface area and electrostatic interactions have been proposed to influence the carrier protein substrate specificity for Mtb's PptT . Given that PptH acts on at least a subset of PptT's substrates, it is reasonable to hypothesize that PptH may bind carrier proteins in a similar manner.…”

“…21,22 Furthermore, the shape of the active-site flanking hydrophobic surface area and electrostatic interactions have been proposed to influence the carrier protein substrate specificity for Mtb's PptT. 5,21 Given that PptH acts on at least a subset of PptT's substrates, 7 it is reasonable to hypothesize that PptH may bind carrier proteins in a similar manner.…”

“…Once activated, these synthases/synthetases carry out biosynthetic processes essential to Mtb' s lipid production, cell wall biosynthesis, and secondary metabolism . AcpS, a type I PPTase, activates the integrated fatty acid synthase (FAS) I carrier protein domain . PptT, the type II PPTase, activates AcpM, the FAS‐II carrier protein, along with the carrier proteins associated with various nonribosomal peptide synthetases and type I polyketide synthases .…”

“…3,5 PptT, the type II PPTase, activates AcpM, the FAS-II carrier protein, along with the carrier proteins associated with various nonribosomal peptide synthetases and type I polyketide synthases. 3,5 Despite the fact that both PPTases activate essential carrier proteins, thus far only PptT has been confirmed to be essential to Mtb in vivo. Leblanc et al established that PptT is essential during both the acute and chronic phases of infection in a mouse model.…”

Decrypting the oscillating nature of the 4′‐phosphopantetheine arm in acyl carrier protein AcpM of Mycobacterium tuberculosis

“…apo‐ACPs namely, apo‐AcpM, apo‐AcpM82 and apo‐EcACP, were transformed to their corresponding crypto‐ACP forms using B. subtilis Sfp, a type II PPTase which is known to activate apo‐AcpM . Loading of NBD probe on apo‐ACP was achieved by following the established protocol of Yin et al .…”