Peter J. Kennelly

Cyanobacterial PPP Family Protein Phosphatases Possess Multifunctional Capabilities and Are Resistant to Microcystin-LR

The structural gene for a putative PPP family protein-serine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820,pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine- and phosphotyrosine-containing proteins and 3-phosphohistidine- and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism.

Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus

A gene, CYP119, for a potential cytochrome P450 has been isolated and sequenced from the extreme acidothermophilic archaeon Sulfolobus solfataricus. The gene predicts a polypeptide of 368 amino acids containing the consensus hemebinding sequence Phe‐Gly‐Xaa‐Gly‐Xaa‐His‐Xaa‐Cys‐Xaa‐Gly‐Xaa3‐Ala‐Arg‐Xaa‐Glu. It most closely resembles the cytochrome P450s found in the bacterium Bacillus subtilis, with which it shares 129 identical amino acid residues (35%). This first sequence of a potential archaeal cytochrome P450 represents an important step in tracing the complex evolutionary history of this biologically important enzyme family.

Protein Ser/Thr/Tyr Phosphorylation in the Archaea

The third domain of life, the Archaea (formerly Archaebacteria), is populated by a physiologically diverse set of microorganisms, many of which reside at the ecological extremes of our global environment. Although ostensibly prokaryotic in morphology, the Archaea share much closer evolutionary ties with the Eukarya than with the superficially more similar Bacteria. Initial genomic, proteomic, and biochemical analyses have revealed the presence of “eukaryotic” protein kinases and phosphatases and an intriguing set of serine-, threonine-, and tyrosine-phosphorylated proteins in the Archaea that may offer new insights into this important regulatory mechanism.

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.

A Phosphohexomutase from the Archaeon Sulfolobus solfataricus Is Covalently Modified by Phosphorylation on Serine

A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N-acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electrostatically interfere with the binding of negatively charged phosphohexose substrates, thus attenuating the catalytic efficiency of the enzyme. Using site-directed mutagenesis, Ser-309 was substituted by aspartic acid to mimic the presence of a phosphoryl group. The V(max) of the mutationally altered protein was only 4% that of the unmodified form. Substitution of Ser-309 with larger, but uncharged, amino acids, including threonine, also decreased catalytic efficiency, but to a lesser extent--three- to fivefold. We therefore predict that phosphorylation of the enzyme in vivo serves to regulate its catalytic activity.

The Membrane-Associated Protein-Serine/Threonine Kinase from Sulfolobus solfataricus Is a Glycoprotein

Treatment of a sodium dodecyl sulfate-polyacrylamide gel with periodic acid-Schiff (PAS) stain or blotting with Galanthus nivalis agglutinin revealed the presence of several glycosylated polypeptides in a partially purified detergent extract of the membrane fraction of Sulfolobus solfataricus. One of the glycoproteins comigrated with the membrane-associated protein-serine/threonine kinase from S. solfataricus, which had been radiolabeled by autophosphorylation with [(32)P]ATP in vitro. Treatment with a chemical deglycosylating agent, trifluoromethanesulfonic acid, abolished PAS staining and reduced the M(r) of the protein kinase from approximately 67,000 to approximately 62,000. Protein kinase activity also adhered to, and could be eluted from, agarose beads containing bound G. nivalis agglutinin. Glycosylation of the protein kinase implies that at least a portion of this integral membrane protein resides on the external surface of the cell membrane.

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates.

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylated exogenous protein substrates. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Kinetic analysis indicated that the K(m) and V(max) values for SynPTP towards p-nitrophenyl phosphate are similar to those of other known bacterial low molecular weight PTPs. Mutagenic alteration of the predicted catalytic cysteine of PTP, Cys(7), to serine abolished enzyme activity. Using a combination of immunodetection, mass spectrometric analysis and mutagenically altered Cys(7)SerAsp(125)Ala-SynPTP, we identified PsaD (photosystem I subunit II), CpcD (phycocyanin rod linker protein) and phycocyanin-α and -β subunits as possible endogenous substrates of SynPTP in this cyanobacterium. These results indicate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803.

The Protein Phosphatases of Synechocystis sp. Strain PCC 6803: Open Reading Frames sll1033 and sll1387 Encode Enzymes That Exhibit both Protein-Serine and Protein-Tyrosine Phosphatase Activity In Vitro

The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp608 by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg2+ or Mn2+, for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.

Inhibition of an archaeal protein phosphatase activity by okadaic acid, microcystin-LR, or calyculin A

Soluble extracts of the methanogenic archaeon, Methanosarcina thermophila TM‐1, contained a divalent metal ion‐stimulated protein‐serine phosphatase activity. This activity was sensitive to micromolar concentrations of okadaic acid, microcystin‐LR, or calyculin A, three compounds thought to be highly specific inhibitors of the type 1/2A/2B genetic superfamily of eukaryotic protein‐serine/threonine phosphatases. The observation that each of these three chemically unrelated compounds inhibited this archaeal protein phosphatase activity suggests the existence of structural homology, and perhaps even common genetic ancestry, with the type 1/2A/2B superfamily of protein‐serine/threonine phosphatases found in eukaryotic organisms.

A PPM-family protein phosphatase from the thermoacidophile Thermoplasma volcanium hydrolyzes protein-bound phosphotyrosine

The genomes of virtually all free-living archaeons encode one or more deduced protein-serine/threonine/tyrosine kinases belonging to the so-called eukaryotic protein kinase superfamily. However, the distribution of their cognate protein-serine/threonine/phosphatases displays a mosaic pattern. Thermoplasma volcanium is unique among the Archaea inasmuch as it is the sole archaeon whose complement of deduced phosphoprotein phosphatases includes a member of the PPM-family of protein-serine/threonine phosphatases—a family that originated in the Eucarya. A recombinant version of this protein, TvnPPM, exhibited protein-tyrosine phosphatase in addition to its predicted protein-serine/threonine phosphatase activity in vitro. TvnPPM is the fourth member of the PPM-family shown to exhibit such dual-specific capability, suggesting that the ancestral versions of this enzyme exhibited broad substrate specificity. Unlike most other archeaons, the genome of T. volcanium lacks open reading frames encoding stereotypical protein-tyrosine phosphatases. Hence, the dual-specificity of TvnPPM may account for its seemingly aberrant presence in an archaeon.