Structure–function relationships in Escherichia coli adenylate cyclase

Abstract

Class I adenylate cyclases are found in γ- and δ-proteobacteria. They play central roles in processes such as catabolite repression in Escherichia coli or development of full virulence in pathogens such as Yersinia enterocolitica and Vibrio vulnificus. The catalytic domain (residues 2–446) of the adenylate cyclase of E. coli was overexpressed and purified. It displayed a Vmax of 665 nmol of cAMP·mg−1·min−1 and a Km of 270 μM. Titration of the metal cofactor Mg2+ against the substrate ATP showed a requirement for free metal ions in addition to the MgATP complex, suggesting a two-metal-ion mechanism as is known for class II and class III adenylate cyclases. Twelve residues which are essential for catalysis were identified by mutagenesis of a total of 20 polar residues conserved in all class I adenylate cyclases. Five essential residues (Ser103, Ser113, Asp114, Asp116 and Trp118) were part of a region which is found in all members of the large DNA polymerase β-like nucleotidyltransferase superfamily. Alignment of the E. coli adenylate cyclase with the crystal structure of a distant member of the superfamily, archaeal tRNA CCA-adding enzyme, suggested that Asp114 and Asp116 are the metal-cofactor-ion-binding residues. The S103A mutant had a 17-fold higher Km than wild-type, demonstrating its important role in substrate binding. In comparison with the tRNA CCA-adding enzyme, Ser103 of the E. coli adenylate cyclase apparently binds the γ-phosphate group of ATP. Consistent with this function, the S103A mutation caused a marked reduction of discrimination between ATP- and ADP- or AMP-derived inhibitors.