Identification of active-site cysteines in the conserved domain of PilD, the bifunctional type IV pilin leader peptidase/N-methyltransferase of Pseudomonas aeruginosa

摘要: PilD is a bifunctional enzyme responsible for cleavage of the leader peptides from precursors type IV pilin and four proteins with pilin-like amino termini that are required extracellular protein secretion in Pseudomonas aeruginosa. Following cleavage, also catalyzes second major posttranslational modification these proteins, namely N-methylation amino-terminal phenylalanine residues mature polypeptides. In this report, we demonstrate enzymatic activities involve cysteine lie within cytoplasmic domain shows high degree similarity to other postulated perform same function bacterial species. Both reduced presence sulfhydryl-reactive reagents such as N-ethylmaleimide p-chloromercuribenzoate. Mutagenesis pilD resulting specific acid substitutions all Cys show 4 conserved cysteines full peptidase activity vivo complete methyltransferase vitro. Conversely, substitution residue membrane spanning had no effect on or Evidence suggests sites adjacent, important methyl donor binding, prior reaction S-adenosyl-L-methionine analogue sinefungin afforded protection inactivation N-ethylmaleimide.