Three distinct families of PTPs, the conventional (cPTPs), low molecular weight (LMW PTPs), and Cdc25 PTPs, have converged upon a common catalytic mechanism and active site sequence, mainly, the phosphate-binding loop encompassing the PTP signature motif (H/V)<b>C</b>(X)₅<b>R</b>(S/T) and an essential Asp residue on a surface loop. There is little sequence similarity among the three families of phosphatases. All known LMW PTP remove phosphoryl groups esterified to the hydroxyl amino acid: tyrosine, whereas all members of the Cdc25 family are dual-specificity protein phosphatases that dephosphorylate all the hydroxyl amino acids: tyrosine, serine and threonine. The cPTP family primarily functions as tyrosine phosphatases, but it also includes dual-specific members.
ORFs encoding potential cPTPs have been identified in five archaeal species: <i>Methanobacterium thermoautotrophicum</i>, <i>Methanococcus jannaschii</i>, <i>Thermococcus kodakaraensis</i>, <i>Pyrococcus horikoshii</i>, and <i>S. solfataricus</i>. Only one has been partially characterized, <i>Tk</i>-PTP from <i>T. kodakaraensis</i>. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented.
The genome of <i>S. solfataricus</i> encodes a single conventional protein tyrosine phosphatase, SsoPTP. SsoPTP is the smallest known archaeal PTP (18.3 kDa) with a primary amino acid sequence that conforms to the cPTP protein tyrosine phosphatase paradigm, HCX₅R(S/T).
Relatively little is known about its mode of action " whether it follows the conventional PTP mechanism or employs a different route for catalysis " or its physiological role.
ORF <i>sso2453</i> from the genome of <i>Sulfolobus solfataricus</i>, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in <i>E. coli</i> and purified by immobilized metal affinity chromatography. SsoPTP displayed the ability to dephosphorylate protein-bound phosphotyrosine as well as protein-bound phosphoserine/phosphothreonine. SsoPTP hydrolyzed both isomers of naphthyl phosphate, an indication of dual specificity. The four conserved residues within the presumed active site sequence: Asp⁶⁹, His⁹⁵, and Arg¹⁰², and the invariant Gln¹³⁹ residue were essential for catalysis, as it was predicted for the established members of the PTP family in both bacteria and eukaryotes. A substrate trapping protein variant, SsoPTP-C96S/D69A, was constructed to isolate possible SsoPTP substrates present in <i>S. solfataricus</i> cell lysates. Several potential substrates were isolated and identified by mass spectroscopy. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/37625 |
| Date | 03 May 2010 |
| Creators | Dahche, Hanan Mohamad |
| Contributors | Biochemistry, Kennelly, Peter J., Li, Jianyong, Helm, Richard F., Bevan, David R. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | Dahche_HM_D_2010.pdf |
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