The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independentlyof its acylation status
T. Basar et al., The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independentlyof its acylation status, J BIOL CHEM, 274(16), 1999, pp. 10777-10783
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.