Title page for ETD etd-04142010-151033

Type of Document Dissertation
Author Dahche, Hanan Mohamad
Author's Email Address hmdahche@vt.edu
URN etd-04142010-151033
Title Dual-specific protein phosphatases in the Archaea
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Kennelly, Peter J. Committee Chair
Bevan, David R. Committee Member
Helm, Richard Frederick Committee Member
Li, Jianyong Committee Member
  • dual-specific phosphatase
  • Archaea
  • protein tyrosine phosphatase
  • protein phosphorylation
Date of Defense 2010-04-02
Availability restricted

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 – whether it follows the conventional PTP mechanism or employs a different route for catalysis – 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.

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