Activation of a cyanobacterial adenylate cyclase, CyaC, by autophosphorylation and a subsequent phosphotransfer reaction

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity, CyaC showed ability to autophosphorylate in vitro with the gamma-phosphate of [gamma-P-32]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His(572)) and receiver domains (Asp(60) or. Asp(895)) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His(572) is the initial phosphorylation site and that the phosphoryl group once bound to His(572) is transferred to Asp(895). The enzyme activities of the CyaC derivatives defective in His572 Or Asp(895) were considerably reduced. Asp(895) is phosphorylated by acetyl [P-32]phosphate, a small phosphoryl molecule, but Asp(60) is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp(895) is intact. These results suggest that the phosphorylation of Asp(895) is essential for the activation of adenylate cyclase and that Asp(60) functions differently from Asp(895) in regulating the enzyme activity.