Structure, biochemistry, and inhibition of essential 4'-phosphopantetheinyl transferases from two species of Mycobacteria

Abstract

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.

Figure 1

Crystal structure of PptT (PDB id 4QJK). β-Sheets are colored orange, and α-helices colored blue. Highlighted side chains are depicted as ball and stick models and colored by element. Ligands are depicted as stick models colored by element with polar hydrogens displayed. (a) Overall structure of PptT complexed with CoA, exhibiting a pseudodimeric structure characteristic of Sfp-type PPTases. (b) The interactions of the pantetheine binding tunnel with CoA are highlighted. The hydrogen bond between the backbone carbonyl oxygen of Leu171 and the amide hydrogen of pantetheine is highlighted with a black dotted line. (c) Depiction of the amino acids that form a deep hydrophobic pocket at the adenine binding site. The phosphopantetheine portion of CoA was omitted for clarity. (d) Coordination of the 3′-phosphate of CoA by Arg56 and Arg48. Lys75 of MuPPT was overlaid onto the PptT structure and depicted as slightly transparent. Depicted in yellow and green are the residues observed in HsPPT (PDB id 2C43) and Sfp (PDB id 1QR0), respectively, which coordinate the 3′-phosphate.

Figure 2

Surface representation of the CoA binding pocket of (a) PptT (PDB id 4QJK) (b) MuPPT (PDB id 4QJL), (c) HsPPT (PDB id 2C43), and (d) Sfp (PDB ids 4MRT, 1QR0). CoA is depicted as color-coded bonds on top of a gray protein-accessible surface. The CoA conformation observed in the 1QR0 Sfp structure is shown as semitransparent bonds, colored with purple carbons, and overlaid on the Sfp and CoA found in 4MRT.

Figure 3

Activity of PPTases. (a) Gels depicting fluorescent labeling of the three carrier protein targets MAS, VibB, and AcpP with rhodamine CoA (mCoA) with PptT. (b) Michaelis–Menten fit of PptT (●), MuPPT (▲), and Sfp (◇) in the BpsA activity assay.