The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independentlyof its acylation status

The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyc lase toxin-hemolysin (ACT) acquires biological activity upon a single amide -linked palmitoylation of the E-amino group of lysine 983 (Lys(983)) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys(860)), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additio nal acylation site by replacing Lys(860) Of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses o f mutant proteins confirmed that the two sites are acylated independently i n vivo and that mutations of Lys860 did not affect the quantitative acylati on of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta 9 C16:1) fatty -acyl groups. Nevertheless, even the most conservative substitution of lysi ne 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a B-fold reduction of cell association capacity and a fu rther 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin, These results suggest that lysine 860 plays by itself a cruci al structural role in membrane insertion and translocation of the toxin, in dependently of its acylation status.