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New page: left|200px<br /><applet load="2ia5" size="450" color="white" frame="true" align="right" spinBox="true" caption="2ia5, resolution 2.90Å" /> '''T4 polynucleotide ki...
 
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'''T4 polynucleotide kinase/phosphatase with bound sulfate and magnesium.'''<br />


==Overview==
==T4 polynucleotide kinase/phosphatase with bound sulfate and magnesium.==
T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of, bifunctional enzymes with 5'-kinase and 3' phosphatase activities that, function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa, polypeptide, which consists of an N-terminal kinase domain of the P-loop, phosphotransferase superfamily and a C-terminal phosphatase domain of the, DxD acylphosphatase superfamily. The homotetramer is formed via pairs of, phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we, identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are, required for 3' phosphatase activity. Alanine mutations at these positions, abolished phosphatase activity without affecting kinase function or, tetramerization. Conservative substitutions of asparagine or glutamate for, Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine, substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in, lieu of Asp277 restored full activity, whereas asparagine at position 277, had no salutary effect. We report a 3.0 A crystal structure of the Pnkp, tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279, in a position that we propose mimics one of the penultimate, phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate., The amalgam of mutational and structural data engenders a plausible, catalytic mechanism for the phosphatase that includes covalent catalysis, (via Asp165), general acid-base catalysis (via Asp167), metal coordination, (by Asp165, Asp277 and Asp278), and transition state stabilization (via, Lys258, Ser211, backbone amides, and the divalent cation). Other critical, side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To, probe the role of oligomerization in phosphatase function, we introduced, six double-alanine cluster mutations at the phosphatase-phosphatase domain, interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from, a tetramer to a dimer and ablated phosphatase activity.
<StructureSection load='2ia5' size='340' side='right'caption='[[2ia5]], [[Resolution|resolution]] 2.90&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[2ia5]] is a 12 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_virus_T4 Escherichia virus T4]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2IA5 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2IA5 FirstGlance]. <br>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.9&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ARS:ARSENIC'>ARS</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=2ia5 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2ia5 OCA], [https://pdbe.org/2ia5 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2ia5 RCSB], [https://www.ebi.ac.uk/pdbsum/2ia5 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2ia5 ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/KIPN_BPT4 KIPN_BPT4] Acts as a 5'-hydroxyl kinase, a 3'-phosphatase and a 2',3'-cyclic phosphodiesterase. Catalyzes the transfer of the terminal phosphate of ATP to the 5'-hydroxyl termini of ribo- and deoxyribonucleotides. In the presence of ADP the enzyme also catalyzes an exchange reaction. In the exchange reaction, an excess ADP causes the enzyme to transfer the 5' terminal phosphate from phosphorylated DNA to ADP. These activities modify the ends of nicked tRNA generated by a bacterial response to infection and facilitate repair by T4 RNA ligase.
== Evolutionary Conservation ==
[[Image:Consurf_key_small.gif|200px|right]]
Check<jmol>
  <jmolCheckbox>
    <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/ia/2ia5_consurf.spt"</scriptWhenChecked>
    <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
    <text>to colour the structure by Evolutionary Conservation</text>
  </jmolCheckbox>
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=2ia5 ConSurf].
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.


==About this Structure==
Structure-function analysis of the 3' phosphatase component of T4 polynucleotide kinase/phosphatase.,Zhu H, Smith P, Wang LK, Shuman S Virology. 2007 Sep 15;366(1):126-36. Epub 2007 May 9. PMID:17493655<ref>PMID:17493655</ref>
2IA5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacteriophage_t4 Bacteriophage t4] with MG, SO4 and ARS as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Polynucleotide_5'-hydroxy-kinase Polynucleotide 5'-hydroxy-kinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.1.78 2.7.1.78] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2IA5 OCA].


==Reference==
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
Structure-function analysis of the 3' phosphatase component of T4 polynucleotide kinase/phosphatase., Zhu H, Smith P, Wang LK, Shuman S, Virology. 2007 May 8;. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17493655 17493655]
</div>
[[Category: Bacteriophage t4]]
<div class="pdbe-citations 2ia5" style="background-color:#fffaf0;"></div>
[[Category: Polynucleotide 5'-hydroxy-kinase]]
== References ==
[[Category: Single protein]]
<references/>
[[Category: Lima, C.D.]]
__TOC__
[[Category: Shuman, S.]]
</StructureSection>
[[Category: Smith, P.C.]]
[[Category: Escherichia virus T4]]
[[Category: Wang, L.K.]]
[[Category: Large Structures]]
[[Category: Zhu, H.]]
[[Category: Lima CD]]
[[Category: ARS]]
[[Category: Shuman S]]
[[Category: MG]]
[[Category: Smith PC]]
[[Category: SO4]]
[[Category: Wang LK]]
[[Category: polynucleotide kinase phosphatase sulfate-complex]]
[[Category: Zhu H]]
 
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