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| [[Image:1kfs.gif|left|200px]]<br /><applet load="1kfs" size="350" color="white" frame="true" align="right" spinBox="true"
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| caption="1kfs, resolution 2.100Å" />
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| '''DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX'''<br />
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| ==Overview== | | ==DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX== |
| A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.
| | <StructureSection load='1kfs' size='340' side='right'caption='[[1kfs]], [[Resolution|resolution]] 2.10Å' scene=''> |
| | == Structural highlights == |
| | <table><tr><td colspan='2'>[[1kfs]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KFS OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1KFS 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]] 2.1Å</td></tr> |
| | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</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=1kfs FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1kfs OCA], [https://pdbe.org/1kfs PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1kfs RCSB], [https://www.ebi.ac.uk/pdbsum/1kfs PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1kfs ProSAT]</span></td></tr> |
| | </table> |
| | == Function == |
| | [https://www.uniprot.org/uniprot/DPO1_ECOLI DPO1_ECOLI] In addition to polymerase activity, this DNA polymerase exhibits 3' to 5' and 5' to 3' exonuclease activity. It is able to utilize nicked circular duplex DNA as a template and can unwind the parental DNA strand from its template. |
| | == 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/kf/1kfs_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=1kfs ConSurf]. |
| | <div style="clear:both"></div> |
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| ==About this Structure== | | ==See Also== |
| 1KFS is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=ZN:'>ZN</scene> and <scene name='pdbligand=MG:'>MG</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KFS OCA].
| | *[[DNA polymerase 3D structures|DNA polymerase 3D structures]] |
| | | __TOC__ |
| ==Reference==
| | </StructureSection> |
| Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates., Brautigam CA, Steitz TA, J Mol Biol. 1998 Mar 27;277(2):363-77. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=9514742 9514742]
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| [[Category: DNA-directed DNA polymerase]]
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| [[Category: Escherichia coli]] | | [[Category: Escherichia coli]] |
| [[Category: Single protein]] | | [[Category: Large Structures]] |
| [[Category: Brautigam, C A.]] | | [[Category: Brautigam CA]] |
| [[Category: Steitz, T A.]] | | [[Category: Steitz TA]] |
| [[Category: MG]]
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| [[Category: ZN]]
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| [[Category: complex (hydrolase/dna)]]
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| [[Category: exonuclease]]
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| [[Category: phosphorothioate]]
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| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:33:40 2008''
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