IDENTIFICATION OF ACTIVE-SITE CYSTEINES IN THE CONSERVED DOMAIN OF PILD, THE BIFUNCTIONAL TYPE-IV PILIN LEADER PEPTIDASE N-METHYLTRANSFERASE OF PSEUDOMONAS-AERUGINOSA

PilD is a bifunctional enzyme responsible for cleavage of the leader p eptides from the precursors of the type IV pilin and four proteins wit h type IV pilin-like amino termini that are required for extracellular protein secretion in Pseudomonas aeruginosa. Following cleavage, PilD also catalyzes the second major post-translational modification of th ese proteins, namely the N-methylation of the amino-terminal phenylala nine residues of the mature polypeptides. In this report, we demonstra te that the enzymatic activities of PilD involve cysteine residues tha t lie within a cytoplasmic domain that shows a high degree of similari ty to other proteins postulated to perform the same function in other bacterial species. Both activities are reduced in the presence of sulf hydryl-reactive reagents such as N-ethylmaleimide and p-chloromercurib enzoate. Mutagenesis of pilD resulting in specific amino acid substitu tions in all of the Cys residues in PilD show that the 4 conserved cys teines in the cytoplasmic domain are required for full peptidase activ ity in vivo and for complete peptidase and methyltransferase activitie s in vitro. Conversely, substitution for a Cys residue in a membrane s panning domain had no effect on PilD activities in vivo or in vitro. E vidence suggests that the peptidase and methyltransferase sites of Pil D are adjacent, with the Cys residues in the cytoplasmic domain import ant for methyl donor binding, as prior reaction of PilD with the S-ade nosyl-L-methionine analogue sinefungin afforded complete protection of peptidase activity from inactivation with N-ethylmaleimide.