Dual-specific protein phosphatases in the Archaea
| dc.contributor.author | Dahche, Hanan Mohamad | en |
| dc.contributor.committeechair | Kennelly, Peter J. | en |
| dc.contributor.committeemember | Li, Jianyong | en |
| dc.contributor.committeemember | Helm, Richard F. | en |
| dc.contributor.committeemember | Bevan, David R. | en |
| dc.contributor.department | Biochemistry | en |
| dc.date.accessioned | 2014-03-14T21:10:30Z | en |
| dc.date.adate | 2010-05-03 | en |
| dc.date.available | 2014-03-14T21:10:30Z | en |
| dc.date.issued | 2010-04-02 | en |
| dc.date.rdate | 2010-05-03 | en |
| dc.date.sdate | 2010-04-14 | en |
| 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)<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. | en |
| dc.description.degree | Ph. D. | en |
| dc.identifier.other | etd-04142010-151033 | en |
| dc.identifier.sourceurl | http://scholar.lib.vt.edu/theses/available/etd-04142010-151033/ | en |
| dc.identifier.uri | http://hdl.handle.net/10919/37625 | en |
| dc.publisher | Virginia Tech | en |
| dc.relation.haspart | Dahche_HM_D_2010.pdf | en |
| dc.rights | In Copyright | en |
| dc.rights.uri | http://rightsstatements.org/vocab/InC/1.0/ | en |
| dc.subject | dual-specific phosphatase | en |
| dc.subject | Archaea | en |
| dc.subject | protein tyrosine phosphatase | en |
| dc.subject | protein phosphorylation | en |
| dc.title | Dual-specific protein phosphatases in the <i>Archaea</i> | en |
| dc.type | Dissertation | en |
| thesis.degree.discipline | Biochemistry | en |
| thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
| thesis.degree.level | doctoral | en |
| thesis.degree.name | Ph. D. | en |
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