Structural and ligand-binding properties of a truncated form of Bacillus anthracis adenylate cyclase and of a catalytically inactive variant in which glutamine substitutes for lysine-346.
A truncated, 541-residue-long, Bacillus anthracis adenylate cyclase was expressed in Escherichia coli. The purified protein (CYA 62) exhibited catalytic and CaM-binding properties identical with those of the wild-type enzyme secreted by B. anthracis. The analysis of the secondary structure of the CYA 62 protein by Fourier transform infrared spectroscopy and circular dichroism revealed the dominance of beta-type structure. The protein shows a relatively low thermal stability with the midpoint denaturation temperature at 45 degrees C. A catalytically inactive variant of CYA 62 in which Gln substituted for Lys-346 (CYA 62 K346Q) was comparatively analyzed for its secondary structure and thermal stability, as well as ligand-binding properties with fluorescent derivatives of ATP and calmodulin. The K346Q variant of CYA 62 has a similar secondary structure and comparable calmodulin binding properties to those of the parent protein and exhibits only slightly reduced thermal stability (the apparent midpoint denaturation temperature is at 43 degrees C). Despite these similarities, the binding of 3'-anthraniloyl-2'-deoxy-ATP (a fluorescent ATP analogue) to the modified protein is severely impaired, from which we conclude that the prime function of Lys-346 in the wild-type enzyme from B. anthracis is to ensure tight binding of the nucleotide substrate to the active site.
Labruyère
E
E
Unité des Antigènes Bactériens, Institut Pasteur, Paris, France.
Mock
M
M
Surewicz
W K
WK
Mantsch
H H
HH
Rose
T
T
Munier
H
H
Sarfati
R S
RS
Bârzu
O
O
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000201:enzymology*; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005973:Glutamine / Q000235:genetics*; D008024:Ligands; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D013050:Spectrometry, Fluorescence; D013054:Spectrophotometry, Atomic; D013055:Spectrophotometry, Infrared; D013696:Temperature
D002147:Calmodulin; D008024:Ligands; D005973:Glutamine; D000262:Adenylyl Cyclases; D008239:Lysine