In order to identify molecular features of the
calmodulin (CaM) activated
adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th
codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long
protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the
recombinant protein are very similar to that of
adenylate cyclase purified from B.
pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of
protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with
trypsin in the presence of CaM converted the
recombinant protein to a 43 kd
protein with no loss of activity; the latter corresponds to the secreted form of B.
pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the
recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an
aspartic acid residue for Trp-242 reduced the affinity of
adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type
protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.
pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the
enzyme.