Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.
Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.
Glaser
P
P
Unité de Régulation de l'Expression Génétique, Institut Pasteur, Paris, France.
Munier
H
H
Gilles
A M
AM
Krin
E
E
Porumb
T
T
Bârzu
O
O
Sarfati
R
R
Pellecuer
C
C
Danchin
A
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000235:genetics*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001886:Bordetella pertussis / Q000201:enzymology / Q000235:genetics*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D002384:Catalysis; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry; D008274:Magnesium; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism
D001427:Bacterial Toxins; D002147:Calmodulin; D005456:Fluorescent Dyes; D062367:ortho-Aminobenzoates; C066294:3'-anthraniloyl-2'-deoxy-ATP; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; D008274:Magnesium