Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis

Owing to the spread of multidrug resistance (MDR) and extensive drug resistance (XDR), there is a pressing need to identify potential targets for the development of more-effective anti-M. tuberculosis (Mtb) drugs. PafA, as the sole Prokaryotic Ubiquitin-like Protein ligase in the Pup-proteasome System (PPS) of Mtb, is an attractive drug target. Here, we show that the activity of purified Mtb PafA is significantly inhibited upon the association of AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride) to PafA residue Serine 119 (S119). Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on the activity of purified Mtb PafA. Structural analysis reveals that although S119 is distant from the PafA catalytic site, it is located at a critical position in the groove where PafA binds the C-terminal region of Pup. Phenotypic studies demonstrate that S119 plays critical roles in the function of Mtb PafA when tested in M. smegmatis. Our study suggests that targeting S119 is a promising direction for developing an inhibitor of M. tuberculosis PafA.