Mass spectral determination of phosphopantetheinylation specificity for carrier proteins in Mycobacterium tuberculosis

Abstract

Phosphopantetheinyl transferases ( PPT ases) are key elements in the modular syntheses performed by multienzyme systems such as polyketide synthases. PPT ases transfer phosphopantetheine derivatives from Coenzyme A to carrier proteins ( CP s), thus orchestrating substrate supply. We describe an efficient mass spectrometry‐based protocol for determining CP specificity for a particular PPT ase in organisms possessing several candidate PPT ases. We show that the CP s MbtL and PpsC, both involved in synthesis of essential metabolites in Mycobacterium tuberculosis , are exclusively activated by the type 2 PPT ase PptT and not the type 1 AcpS. The assay also enables conclusive identification of the reactive serine on each CP .

CoA‐dependent activation of mitochondrial acyl carrier protein links four neurodegenerative diseases

“…For this, we chose Drosophila melanogaster because of its conserved metabolic steps and genes and its versatile genetic tools. mtACP requires activation in order to function; the active holo form is generated by enzymatic transfer of a negatively charged 4′‐phosphopantetheine moiety to a conserved serine residue of the inactive apo form (Elovson & Vagelos, ; Jung et al , ; Fig A). For our study we manipulated mtacp , the Drosophila melanogaster gene encoding mtACP, containing Ser‐99, which is predicted to bind 4‐phosphopantetheine (Ragone et al , ).…”

A combination of docking and cheminformatics approaches for the identification of inhibitors against 4′ phosphopantetheinyl transferase ofMycobacterium tuberculosis

“…Based on the docking studies, a total of 205 top scoring molecules, named as P-1 to P-205, were procured from NCI/DTP and [10][11][12][13] and thus have carried out crystallization and enzyme characterization studies by using MBP-PptT fusion protein. 8,12,13 Additionally, these studies have also reported that cleavage of MBP tag results in signicant precipitation of PptT protein thereby making it difficult to work with native PptT. 10,12,13 Thus, we used MBP-PptT fusion protein for our activity assays and inhibition studies with M. tb AcpM as the substrate.…”

“…8,12,13 Additionally, these studies have also reported that cleavage of MBP tag results in signicant precipitation of PptT protein thereby making it difficult to work with native PptT. 10,12,13 Thus, we used MBP-PptT fusion protein for our activity assays and inhibition studies with M. tb AcpM as the substrate. In order to screen 205 molecules, we rst characterized PptT by carrying out the time kinetics as well as K m experiment with varying concentrations of CoA by using UPLC based method for precise quantication of apo AcpM and holo AcpM peaks.…”

“…Also, Jung et al demonstrated specic activation of carrier proteins MbtL and PpsC via PptT and not via AcpS. 12 Thus, various genetic and biochemical studies supported the non-redundant as well as crucial role of PptT in M. tb. Further, the elucidation of 3dimensional crystal structures of PptT as MBP-PptT fusion protein 13 as well as the native PptT 14 provided insights into the key residues involved in the binding of coenzyme A.…”

“…Also, various researchers biochemically demonstrated the non-redundant nature of these two proteins by demonstrating the activation of a specic set of proteins only by PptT and not by AcpS. 11,12 Zimhony et al 11 showed that AcpM, an important acyl carrier protein of M. tb involved in mycolic acid synthesis, was activated by PptT and not by AcpS. Also, Jung et al demonstrated specic activation of carrier proteins MbtL and PpsC via PptT and not via AcpS.…”