Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus.
When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [gamma-(32)P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of approximately 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 Co(2+) or Mn(2+). The recombinant protein underwent autophosphorylation when incubated with either [gamma-(32)P]ATP or [gamma-(32)P]GTP. The site of phosphorylation was identified as Ser(59), 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 (32)P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of (32)P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.
Potters
M Ben
MB
Department of Biochemistry and Virginia Institute for Genomics, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Solow
Barbara T
BT
Bischoff
Kenneth M
KM
Graham
David E
DE
Lower
Brian H
BH
Helm
Richard
R
Kennelly
Peter J
PJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003035:Cobalt / Q000378:metabolism; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010736:Phosphoglycerate Mutase / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
D019843:Archaeal Proteins; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; D012694:Serine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010736:Phosphoglycerate Mutase