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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 peptides from the precursors of the type IV pilin and four proteins with type IV pilin-like amino termini that are required for extracellular protein secretion in Pseudomonas aeruginosa. Following cleavage, PilD also catalyzes the...
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Published in: | The Journal of biological chemistry 1993-07, Vol.268 (21), p.15788-15794 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | PilD is a bifunctional enzyme responsible for cleavage of the leader peptides from the precursors of the type IV pilin and
four proteins with type IV pilin-like amino termini that are required for extracellular protein secretion in Pseudomonas aeruginosa.
Following cleavage, PilD also catalyzes the second major posttranslational modification of these proteins, namely the N-methylation
of the amino-terminal phenylalanine residues of the mature polypeptides. In this report, we demonstrate that the enzymatic
activities of PilD involve cysteine residues that lie within a cytoplasmic domain that shows a high degree of similarity to
other proteins postulated to perform the same function in other bacterial species. Both activities are reduced in the presence
of sulfhydryl-reactive reagents such as N-ethylmaleimide and p-chloromercuribenzoate. Mutagenesis of pilD resulting in specific
amino acid substitutions in all of the Cys residues in PilD show that the 4 conserved cysteines in the cytoplasmic domain
are required for full peptidase activity in vivo and for complete peptidase and methyltransferase activities in vitro. Conversely,
substitution for a Cys residue in a membrane spanning domain had no effect on PilD activities in vivo or in vitro. Evidence
suggests that the peptidase and methyltransferase sites of PilD are adjacent, with the Cys residues in the cytoplasmic domain
important for methyl donor binding, as prior reaction of PilD with the S-adenosyl-L-methionine analogue sinefungin afforded
complete protection of peptidase activity from inactivation with N-ethylmaleimide. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)82324-9 |