Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

Abstract

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.

• Authors: P Glaser, A Elmaoglou-Lazaridou, E Krin, D Ladant, O Bârzu, A Danchin
• Journal: The EMBO journal (EMBO J) Vol. 8 Issue 3 Pg. 967-72 (Mar 1989) ISSN: 0261-4189 [Print] England
• PMID: 2542030 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Calmodulin
• Recombinant Fusion Proteins
• Adenylyl Cyclases

Topics

• Adenylyl Cyclases (genetics, metabolism)
• Amino Acid Sequence
• Binding Sites
• Bordetella pertussis (genetics, metabolism)
• Calmodulin (metabolism)
• Cloning, Molecular
• Escherichia coli (genetics)
• Kinetics
• Molecular Sequence Data
• Mutation
• Recombinant Fusion Proteins (genetics, metabolism)