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Journal of Bacteriology, April 2003, p. 2112-2121, Vol. 185, No. 7
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.7.2112-2121.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon Sulfolobus solfataricus

M. Ben Potters, Barbara T. Solow, Kenneth M. Bischoff, David E. Graham, Brian H. Lower, Richard Helm, and Peter J. Kennelly*

Department of Biochemistry and Virginia Institute for Genomics, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

Received 9 August 2002/ Accepted 30 December 2002

When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [{gamma}-32P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of ~46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co2+ or Mn2+. The recombinant protein underwent autophosphorylation when incubated with either [{gamma}-32P]ATP or [{gamma}-32P]GTP. The site of phosphorylation was identified as Ser59, which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the 32P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of 32P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.

* Corresponding author. Mailing address: Department of Biochemistry-0308, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. Phone: (540) 231-4317. Fax: (540) 231-9070. E-mail: pjkennel{at}vt.edu.

Journal of Bacteriology, April 2003, p. 2112-2121, Vol. 185, No. 7
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.7.2112-2121.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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