4′-Phosphopantetheinyl
transferases (PPTase) post-translationally
modify carrier proteins with a phosphopantetheine moiety, an essential
reaction in all three domains of life. In the bacterial genus Mycobacteria, the Sfp-type PPTase activates pathways necessary
for the biosynthesis of cell wall components and small molecule virulence
factors. We solved the X-ray crystal structures and biochemically
characterized the Sfp-type PPTases from two of the most prevalent
Mycobacterial pathogens, PptT of M. tuberculosis and
MuPPT of M. ulcerans. Structural analyses reveal
significant differences in cofactor binding and active site composition
when compared to previously characterized Sfp-type PPTases. Functional
analyses including the efficacy of Sfp-type PPTase-specific inhibitors
also suggest that the Mycobacterial Sfp-type PPTases can serve as
therapeutic targets against Mycobacterial infections.
The discovery of peptide substrates for enzymes with exclusive, selective activities is a central goal in chemical biology. In this paper, we develop a hybrid computational and biochemical method to rapidly optimize peptides for specific, orthogonal biochemical functions. The method is an iterative machine learning process by which experimental data is deposited into a mathematical algorithm that selects potential peptide substrates to be tested experimentally. Once tested, the algorithm uses the experimental data to refine future selections. This process is repeated until a suitable set of de novo peptide substrates are discovered. We employed this technology to discover orthogonal peptide substrates for 4’-phosphopantetheinyl transferase, an enzyme class that covalently modifies proteins. In this manner, we have demonstrated that machine learning can be leveraged to guide peptide optimization for specific biochemical functions not immediately accessible by biological screening techniques, such as phage display and random mutagenesis.
The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins. Here we demonstrate the full reversibility of post-translational custom pantetheine modification of E. coli acyl carrier protein (ACP) for visualization and functional studies. We utilize this iterative enzymatic methodology in vitro for reversible labeling variants and apply these tools to Nuclear Magnetic Resonance (NMR) structural studies of protein-substrate interactions.
4′-Phosphopantetheinyl
transferases (PPTases) catalyze a post-translational modification
essential to bacterial cell viability and virulence. We present the
discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar
inhibition of bacterial Sfp-PPTase with no activity toward the human
orthologue. Moreover, compounds within this class possess antibacterial
activity in the absence of a rapid cytotoxic response in human cells.
An advanced analogue of this series, ML267 (55), was
found to attenuate production of an Sfp-PPTase-dependent metabolite
when applied to Bacillus subtilis at
sublethal doses. Additional testing revealed antibacterial activity
against methicillin-resistant Staphylococcus aureus, and chemical genetic studies implicated efflux as a mechanism for
resistance in Escherichia coli. Additionally,
we highlight the in vitro absorption, distribution, metabolism, and
excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule
inhibitor.
Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity.
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