Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.
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.
Glaser
P
P
Unité de Régulation de l'Expression Génétique, Institut Pasteur, Paris, France.
Elmaoglou-Lazaridou
A
A
Krin
E
E
Ladant
D
D
Bârzu
O
O
Danchin
A
A
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001886:Bordetella pertussis / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D000262:Adenylyl Cyclases