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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.

ATP hydrolysis and synthesis by the F 0 F 1 -ATP synthase are coupled to proton translocation across the membrane in the presence of magnesium. Calcium is known, however, to disrupt this coupling in the photosynthetic enzyme in a unique way: it does not support ATP synthesis, and CaATP hydrolysis is decoupled from any proton translocation, but the membrane does not become leaky to protons. Understanding the molecular basis of these calcium-dependent effects can shed light on the as yet unclear mechanism of coupling between proton transport and rotational catalysis. We show here, using an actin filament ␥-rotation assay, that CaATP is capable of sustaining rotational motion in a highly active hybrid photosynthetic F 1 -ATPase consisting of ␣ and ␤ subunits from Rhodospirillum rubrum and ␥ subunit from spinach chloroplasts (␣ Recent experiments have provided strong evidence for the rotational catalysis mechanism proposed for the ubiquitous enzyme F 0 F 1 -ATPase/ATP synthase (1, 2). Particularly striking is the effort led by several laboratories to measure the rotational motion of the F 1 part of the enzyme on the singlemolecule level. F 1 is constructed of five subunits, with a stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. Fluorescent actin filaments attached to the ␥ subunit of engineered surface-immobilized ␣ 3 ␤ 3 ␥ subcomplexes of the enzyme, either from a thermophyilic bacterium, TF 1 1 (3), or from Escherichia coli, EF 1 (4, 5), were shown to rotate unidirectionally while hydrolyzing MgATP. This rotation was not only dependent on ATP concentration but was shown to have almost 100% efficiency of chemical to mechanical energy conversion. At very low ATP concentration the stepwise motion of the enzyme was exposed (6). Small beads attached to ␥ in place of the actin filament allowed Yasuda et al.(7) to follow the motion at the limit of small load and watch rotation at a rate as high as 130 revolutions per second. Most spectacularly, forced clockwise rotation was shown very recently to lead to ATP synthesis, thus confirming the essential role of ␥ subunit rotation in both directions of the reaction (8). Some single-molecule work also addressed the operation of the F 0 part of the F 0 F 1 -ATP synthase. It was shown that the F 0 -c subunit oligomer, which consists of 10 -14 identical c subunits, is capable of rotating together with ␥ (9, 10).While much new information has been collected regarding rotational catalysis in the ATP synthase, there are still many open questions, mainly in relation to the coupling between enzymatic activity and rotational motion. The photosynthetic version of the ATP synthase is an attractive system to probe some of these questions, since it presents several distinct functional properties. These include the tight regulation of ATP hydrolysis, which is especially important in photosynthetic cells, where it prevents the depletion of essential ATP pools in the dark (11-13). Indeed, plant chloroplasts have a unique regulatory system, termed thiol modulation, which leads to reduction of a disulfide bond fo...

BackgroundFatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS).MethodsIn order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography.ResultsActivation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM.ConclusionPurified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

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