Activation of a Cyanobacterial Adenylate Cyclase, CyaC, by Autophosphorylation and a Subsequent Phosphotransfer Reaction*
- From the Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153, Japan
Abstract
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 inEscherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylatein vitro with the γ-phosphate of [γ-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) 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 His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity.
Footnotes
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↵* This work was supported by a Grant-in-aid for General Scientific Research (07404045) from the Ministry of Education, Science, Sports and Culture of Japan (to M. O.) and also by a grant from Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (to M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153, Japan. Tel.: 81-3-5454-6631; Fax: 81-3-5454-4333; E-mail: cohmori{at}komaba.ecc.u-tokyo.ac.jp.
- Abbreviations:
- PCR
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polymerase chain reaction
- kb
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kilobase pair(s)
- PMSF
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phenylmethylsulfonyl fluoride
- DTT
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dithiothreitol
- PAGE
-
polyacrylamide gel electrophoresis
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- Received December 22, 1998.
- Revision received February 26, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
