Figure 1.
Schematic representation of phosphopantetheinyl transferase (PPTase) catalyzed conversion of apo-ACP to holo-ACP.
Phosphopantetheine group of coenzyme A is transferred by the PPTases to a conserved serine group of ACP. This post-translational modification of carrier proteins is necessary for their function in various metabolic pathways.
Figure 2.
Phylogenetic analysis and expression profile of PPTases.
A, Sequences of PPTases from Dictyostelium and other lower eukaryotes were analyzed for their evolutionary relatedness. The sequences branch into two distinct groups, one group constituting AcpS-like sequences and the other formed by Sfp-like sequences. B, Expression profiles of DiAcpS and DiSfp were studied by RT-PCR and both were found to be expressing at all developmental stages. Stages analyzed were amoeboid (1), 0 hrs. after starvation (2), streaming (3), loose aggregate (4), mound (5), slug (6), early culminant (7) and fruiting body (8). IG7 (mitochondrial large rRNA) was used as the RT-PCR control.
Figure 3.
Differential specificity of Dictyostelium Sfp (DiSfp) towards mycobacterial PKS2 and kinetic analysis.
Gel-binding assays were set up with DiAcpS and DiSfp, taking [1-14C] acetyl-CoA (A-CoA), [1-14C] hexanoyl-CoA (H-CoA) and [1-14C] lauroyl-CoA (L-CoA) as co-substrates. A, Panels show the autoradiogram of SDS-PAGE in which mycobacterial PKS2 was labeled by different acyl CoA substrates and PPTases as indicated. B, Gel-based kinetic analysis of DiSfp with respect to acetyl CoA. Concentration of acetyl CoA was varied from 5 µM to 45 µM.
Figure 4.
Differential specificity of Dictyostelium AcpS (DiAcpS) towards mycobacterial stand-alone ACP and kinetic analysis.
A, Panels show the autoradiogram of SDS-PAGE in which mycobacterial Rv1344 was labeled by different acyl CoA substrates and PPTases as indicated. Rv1344 shows the presence of two protein bands, lower one being the N-terminus truncated form. The truncated form is also seen to be incorporating radioactivity, suggestive of PPTase mediated modification. B, Michaelis-Menten kinetic analysis of DiAcpS was performed in a similar way as that for DiSfp.
Figure 5.
Rv1344 phosphopantetheinylation by DiAcpS.
A, HPLC chromatogram of assays with DiAcpS. B, HPLC chromatogram for DiSfp reactions. Reactions were carried out with coenzyme A (peak 2), hexanoyl CoA (peak 3) and lauroyl CoA (peak 4). Peak 1 represents apo-form of the ACP in each case. Each peak was subjected to MALDI-TOF analysis and the molecular masses obtained for each peak have been indicated.
Figure 6.
Modification of independent ACP domain of mycobacterial PKS12.
Autoradiography shows acetyl-phosphopantetheinylation of the independent ACP domain of multi-functional PKS by DiSfp. DiAcpS is unable to incorporate radioactivity into the ACP.
Figure 7.
Activity of DiAcpS and DiSfp towards Dictyostelium multi-functional ACP and stand-alone ACP.
Gel-binding assays were performed with radioactive acetyl CoA (A-CoA) to confirm the specificities of the PPTases with cognate ACPs. A, ACP-TypeIII PKS di-domain of DiPKS1 shows radiolabeling with DiSfp, but not with DiAcpS. B, ACP domain of DiPKS16 is also modified just by DiSfp. C, the stand-alone ACP, DDB0184099, shows converse pattern. DiAcpS is able to mediate the conversion of apo-form of this ACP to holo-form and DiSfp fails to show activity.
Figure 8.
HPLC-MALDI assays for assessing DiSfp and DiAcpS activity towards DiPKS16 ACP domain and stand-alone ACP respectively.
A, HPLC chromatogram of DiSfp reactions with DiPKS16 ACP. Reactions with coenzymeA, hexanoyl CoA and lauroyl CoA are illustrated as peaks 2, 3 and 4 respectively. Peak 1 in each case corresponds to apo-ACP. B, MALDI-TOF analysis of the peaks obtained in Fig. 8A is depicted. C, HPLC traces of DiAcpS reactions with Dictyostelium stand-alone ACP are represented. D, MALDI-TOF analysis of the peaks obtained in Fig. 8C is shown.