The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene.
In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA(Glc), a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA(Glc), cAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA(Glc). In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA(Glc) were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA(Glc). In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.
Crasnier
M
M
Unité de Régulation de l'Expression Génétique (Centre National de la Recherche Scientifique Unité Associée 1129), Institut Pasteur, Paris, France.
Dumay
V
V
Danchin
A
A
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D000242:Cyclic AMP / Q000096:biosynthesis*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D000262:Adenylyl Cyclases; D005947:Glucose