Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus

Abstract

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.

FIG. 1.

Identification of site of phosphorylation on rSso417 by mass spectrometry. rSso0417 was incubated with ATP, isolated by SDS-PAGE, and hydrolyzed into peptides by using trypsin. These peptides were then analyzed by LC-MS and LC-MS-MS as described in Materials and Methods. Shown is the sequence of the phosphate containing peptide, and below it the MS² spectra of the [M + 2H⁺]²⁺ ion (average m/z = 1,941.6) used to determine the position of the serine residue phosphorylated (pS) with the major ions and the sites of collisional-induced dissociation that produced them indicated. The peak resulting from neutral loss of phosphate is designated [M + 2H⁺ − H₃PO₄]²⁺.

FIG. 2.

Comparison of DNA-derived amino acid sequence of Sso0417 with archaeal, bacterial, and eukaryotic iPGMs. Shown is the DNA-derived amino acid sequence of the protein product of ORF sso0417 from S. solfataricus (Sso0417 [38], GenBank accession no. Q980A0) and, immediately below it, the sequence of iPGMs from the archaeon P. furiosus (Pfs iPGM [42]; GenBank accession no. AAL82083), the eukaryote T. brucei (Tbr iPGM [8]; GenBank accession no. CAB85498), and the bacterium B. stearothermophilus (Bst iPGM [6]; GenBank accession no. AF120091). The sequences were aligned by using the CLUSTALW program available from the European Bioinformatics Institute, with the C-terminal ca. 60 residues adjusted by eye. Amino acid identities between each of the established iPGMs and Sso0417 are indicated by colons, whereas nonidentical amino acids of very similar character are indicated by periods. Amino acids comprising subdomain A of iPGM from B. stearothermophilus (26) are underlined. Amino acids involved in metal ion binding in iPGM are marked by asterisks. Other amino acid residues that are conserved among the subdomain B regions of bacterial and eucaryal iPGMs are indicated by plus signs. The catalytically essential serine residue of the iPGM from B. stearothermohpilus is marked by “@.”

FIG. 3.

rSso0417 possesses PGA mutase activity. The PGA mutase activity of both rSso0417 (•) and a mutationally altered form in which Ser₅₉ was altered to Thr (○) was measured spectrophotometrically by using a coupled assay system that measures the increase in absorbance at 240 nm that takes place when 2-PGA is converted to phosphoenolpyruvate by enolase. Also shown are the changes in absorbance observed in control assays in which either the potential PGM (□), enolase (▵), or 3-PGA (▪) was omitted. All assays were performed in triplicate, with error bars indicating the magnitude of the standard error. For further details, see Materials and Methods.

FIG. 4.

Metal ion preference of rSso0417. rSso0417 was phosphorylated with [γ-³²P]ATP to a stoichiometry of ∼0.5 mol/mol, purified, and then dialyzed against buffer containing 0.05 mM EDTA to remove any remaining metals. The phosphoenzyme was incubated with one of the metal ions listed, each at a concentration of 0.1 mM, for 5 min at a temprature of 65°C in the presence or absence of 3-PGA at 0.1 mM. The protein was then isolated by SDS-PAGE, and the amount of protein-bound [³²P]phosphate determined by electronic autoradiography. The control represents enzyme that was incubated for 5 min at 65°C in the absence of both metals and 3-PGA. In prior experiments it was observed that incubation with 3-PGA in the absence of metals had no effect on enzyme-bound phosphate. For further details, see Materials and Methods.