<p>Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that synthesise bioactive peptides using an assembly line architecture, wherein each module is responsible for the incorporation of a monomer into the growing chain. Present in both fungi and bacteria, NRPSs are responsible for a wide variety of secondary metabolites and bioactive compounds including siderophores, antibiotics, anti-cancer compounds and immunosuppressants. For functionality, NRPSs require the attachment of a phosphopantetheine moiety to their peptidyl carrier protein domains. This reaction is catalysed by a phosphopantetheinyl transferase (PPTase). The NRPS blue pigment synthetase A (BpsA) is unusual in that it is comprised of only a single module. BpsA contains an adenylation domain that recognises and sequentially binds two molecules of L-glutamine, an oxidation domain that is believed to oxidise each glutamine monomer, a peptidyl carrier protein domain that binds the phosphopantetheine moiety, and a thioesterase domain that cyclises each glutamine and releases the final bicyclic product from the enzyme. This final product is a blue pigment called indigoidine, and its synthesis from two molecules of L-glutamine is powered by ATP. Comparatively to other NRPSs BpsA is easy to manipulate and work with both in vitro and in vivo. Here, the ability to easily detect synthesis of indigoidine was utilised to provide a versatile reporter to detect a variety of biochemical activities. PPTases are essential enzymes that are promising drug targets in the clinically important bacteria Pseudomonas aeruginosa and Mycobacterium tuberculosis. BpsA can be purified in the inactive apo form, which then requires a PPTase to activate it to enable indigoidine synthesis. Here it was shown that mixing BpsA, a PPTase, the enzymatic substrates, and a potential inhibitor enables screening for PPTase inhibition by monitoring the rate or extent of indigoidine synthesis. This method was optimised and used to screen commercial drug libraries against two PPTases, PcpS from P. aeruginosa and PptT from M. tuberculosis. Several novel inhibitors were identified and pilot in vivo studies were performed. M. tuberculosis also possesses a second essential PPTase called TB-AcpS, which has very narrow substrate specificity and cannot post-translationally modify BpsA. In an attempt to widen the substrate specificity a combination of rational engineering and directed evolution was employed. These attempts did not yield significant improvements in the ability of TB-AcpS to activate modified BpsA, however they did yield mutants that were more effective substrates for other type I PPTases. The easily detectable nature of indigoidine also enabled application of BpsA as a reporter for a range of different substrates. Particularly effective was development of a commercially applicable method using BpsA to quantify L-glutamine in a range of conditions, including cell culture media and blood. The assay developed offers several advantages over currently available kits. BpsA was also used to detect and quantify ATP, and this was applied to monitor adenylation reactions. Finally, the ability of BpsA to synthesise indigoidine-like compounds from glutamine analogues was explored.</p>
Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT
