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dc.contributor.authorDahche, Hanan Mohamaden_US
dc.date.accessioned2014-03-14T21:10:30Z
dc.date.available2014-03-14T21:10:30Z
dc.date.issued2010-04-02en_US
dc.identifier.otheretd-04142010-151033en_US
dc.identifier.urihttp://hdl.handle.net/10919/37625
dc.description.abstract

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)C(X)5R(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: Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Thermococcus kodakaraensis, Pyrococcus horikoshii, and S. solfataricus. Only one has been partially characterized, Tk-PTP from T. kodakaraensis. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented.

The genome of S. solfataricus 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 â whether it follows the conventional PTP mechanism or employs a different route for catalysis â or its physiological role.

ORF sso2453 from the genome of Sulfolobus solfataricus, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in E. coli 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, Cys96, 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 S. solfataricus cell lysates. Several potential substrates were isolated and identified by mass spectroscopy.

en_US
dc.publisherVirginia Techen_US
dc.relation.haspartDahche_HM_D_2010.pdfen_US
dc.rightsI hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Virginia Tech or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.en_US
dc.subjectdual-specific phosphataseen_US
dc.subjectArchaeaen_US
dc.subjectprotein tyrosine phosphataseen_US
dc.subjectprotein phosphorylationen_US
dc.titleDual-specific protein phosphatases in the Archaeaen_US
dc.typeDissertationen_US
dc.contributor.departmentBiochemistryen_US
dc.description.degreePh. D.en_US
thesis.degree.namePh. D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplineBiochemistryen_US
dc.contributor.committeechairKennelly, Peter J.en_US
dc.contributor.committeememberLi, Jianyongen_US
dc.contributor.committeememberHelm, Richard Fredericken_US
dc.contributor.committeememberBevan, David R.en_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-04142010-151033/en_US
dc.date.sdate2010-04-14en_US
dc.date.rdate2010-05-03
dc.date.adate2010-05-03en_US


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