PMID:2108958

Citation	Xia, ZG and Storm, DR (1990) A-type ATP binding consensus sequences are critical for the catalytic activity of the calmodulin-sensitive adenylyl cyclase from Bacillus anthracis. J. Biol. Chem. 265:6517-20
Abstract	Analysis of the predicted amino acid sequence of Bacillus anthracis adenylyl cyclase revealed sequences with homology to consensus sequences for A- and B-type ATP binding domains found in many ATP binding proteins. Based on the analysis of nucleotide binding proteins, a conserved basic amino acid residue in the A-type consensus sequence and a conserved acidic amino acid residue in the B-type consensus sequence have been implicated in the binding of ATP. The putative ATP binding sequences in the B. anthracis adenylyl cyclase possess analogous lysine residues at positions 346 and 353 within two A-type consensus sequences and a glutamate residue at position 436 within a B-type consensus sequence. The two A-type consensus sequences overlap each other and have the opposite orientation. To determine whether Lys-346, Lys-353, or Glu-436 of the B. anthracis adenylyl cyclase are crucial for enzyme activity, Lys-346 and Lys-353 were replaced with methionine and Glu-436 with glutamine by oligonucleotide-directed mutagenesis. Furthermore, Lys-346 was also replaced with arginine. The genes encoding the wild type and mutant adenylyl cyclases were placed under the control of the lac promoter for expression in Escherichia coli, and extracts were assayed for adenylyl cyclase activity. In all cases, a 90-kDa polypeptide corresponding to the catalytic subunit of the enzyme was detected in E. coli extracts by rabbit polyclonal antibodies raised against the purified B. anthracis adenylyl cyclase. The proteins with the Lys-346 to methionine or arginine mutations exhibited no adenylyl cyclase activity, indicating that Lys-346 in the A-type ATP binding consensus sequence plays a critical role for enzyme catalysis. Furthermore, the enzyme with the Lys-353 to methionine mutation was also inactive, suggesting that Lys-353 may also directly contribute to enzyme catalysis. In contrast, the protein with the Glu-436 to glutamine mutation retained 75% of enzyme activity, suggesting that Glu-436 in the B-type ATP binding consensus sequence may not be directly involved in enzyme catalysis. It is concluded that Lys-346 and Lys-353 in B. anthracis adenylyl cyclase may interact directly with ATP and contribute to the binding of the nucleotide to the enzyme.
Links	PubMed
Keywords	Adenosine Triphosphate/metabolism; Adenylate Cyclase/genetics; Adenylate Cyclase/metabolism; Amino Acid Sequence; Bacillus anthracis/enzymology; Bacillus anthracis/genetics; Base Sequence; Binding Sites; Calmodulin/pharmacology; Cloning, Molecular; Escherichia coli/genetics; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Plasmids
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BACAN:CYAA	enables	GO:0008294: calcium- and calmodulin-responsive adenylate cyclase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
BACAN:CYAA		GO:0008294: calcium- and calmodulin-responsive adenylate cyclase activity	ECO:0000314:		F		Table 1: Adenylyl cyclase activity corrected for the amount of recombinant protein expressed.	complete CACAO 10499
BACAN:CYAA		GO:0008237: metallopeptidase activity	ECO:0000314:		F		See section 'Activation of EF3 by Mutant Forms of CaM', which discusses the activity of protein mutants that have some of the four standard Ca2+ altered.	complete CACAO 10512

References

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