Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase - Substitutions of alanine 140 modulate acylation site selectivity of the toxin acyltransferase CyaC

Citation
T. Basar et al., Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase - Substitutions of alanine 140 modulate acylation site selectivity of the toxin acyltransferase CyaC, J BIOL CHEM, 276(1), 2001, pp. 348-354
Citations number
46
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
1
Year of publication
2001
Pages
348 - 354
Database
ISI
SICI code
0021-9258(20010105)276:1<348:AOL9IS>2.0.ZU;2-E
Abstract
The capacity of adenylate cyclase toxin (ACT) to penetrate into target cell s depends on post-translational fatty-acylation by the acyltransferase CyaC , which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, th e in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic se rine 20 and histidine 33 residues ablated acylating activity of CyaC, Conse rvative replacements of alanine 140 by glycine (A140G) and valine (A140V) r esidues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983 , or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost excl usively at Lys-983, The respective proportion of toxin molecules acylated a t Lye-983 correlated well with the cell-invasive activity of both ACT mixtu res, which was about half of that of ACT fully acylated on Lys-983 by intac t CyaC, These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary an d sufficient.