Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase.
The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient lon- E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s-1 at 30 degrees C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and N-chlorosuccinimide. We show that the calmodulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase.
Labruyère
E
E
Unité des Antigènes Bactériens, Institut Pasteur, Paris, France.
Mock
M
M
Ladant
D
D
Michelson
S
S
Gilles
A M
AM
Laoide
B
B
Bârzu
O
O
D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002872:Chromosome Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D010777:Photochemistry
D000345:Affinity Labels; D002147:Calmodulin; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases