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1qqq

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(New page: 200px<br /><applet load="1qqq" size="450" color="white" frame="true" align="right" spinBox="true" caption="1qqq, resolution 1.50&Aring;" /> '''CRYSTAL STRUCTURE AN...)
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[[Image:1qqq.gif|left|200px]]<br /><applet load="1qqq" size="450" color="white" frame="true" align="right" spinBox="true"
 
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caption="1qqq, resolution 1.50&Aring;" />
 
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'''CRYSTAL STRUCTURE ANALYSIS OF SER254 MUTANT OF ESCHERICHIA COLI THYMIDYLATE SYNTHASE'''<br />
 
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==Overview==
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==CRYSTAL STRUCTURE ANALYSIS OF SER254 MUTANT OF ESCHERICHIA COLI THYMIDYLATE SYNTHASE==
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Drug-resistant variants of thymidylate synthase (TS) can potentially be, used in gene therapy applications to decrease the myelosuppressive side, effects of TS-directed anticancer agents or to select genetically modified, cells in vivo. Mutations of proline 303 of human TS confer resistance to, TS-directed fluoropyrimidines and antifolates (). We generated the, corresponding variants in Escherichia coli TS (ecTS), position 254, to, better understand the mechanism by which mutations at this residue confer, resistance. In addition, because ecTS is intrinsically resistant to, several antifolates when compared with human TS, we suspected that greater, resistance could be achieved with the bacterial enzyme. The P254L enzyme, conferred &gt;100-fold resistance to both raltitrexed and, 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four, additional mutants (P254F, P254S, P254G, and P254D), each of which, complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were, not substantially different among the variants and were comparable with, the wild-type values, but K(m) for methylenetetrahydrofolate, (CH(2)H(4)PteGlu) was &gt;10-fold higher for P254D. Values of k(on) and, k(off) for nucleotide binding, which were obtained by stopped-flow, spectroscopy, were virtually unchanged among the mutants. Drastic, differences were observed for CH(2)H(4)PteGlu binding, with K(d) values, &gt;15-fold higher than observed with the wild-type enzyme; surprisingly, the, proposed isomerization reaction that is very evident for the wild-type, enzyme is not observed with P254S. The decrease in affinity for, CH(2)H(4)PteGlu correlates well with K(i) values obtained for three, TS-directed inhibitors. These results show that mutations at Pro-254, specifically affect the initial binding interactions between enzyme and, cofactor and also alter the ability of the mutant enzymes to undergo, conformational changes that occur on ternary complex formation. The, crystal structure of P254S was determined at 1.5 A resolution and is the, most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253;, its carbonyl is rotated approximately 40 degrees, and the side chain forms, an ion pair with Arg-225.
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<StructureSection load='1qqq' size='340' side='right'caption='[[1qqq]], [[Resolution|resolution]] 1.50&Aring;' scene=''>
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== Structural highlights ==
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<table><tr><td colspan='2'>[[1qqq]] is a 1 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=1QQQ OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1QQQ FirstGlance]. <br>
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</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.5&#8491;</td></tr>
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<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CME:S,S-(2-HYDROXYETHYL)THIOCYSTEINE'>CME</scene>, <scene name='pdbligand=CXM:N-CARBOXYMETHIONINE'>CXM</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr>
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<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1qqq FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1qqq OCA], [https://pdbe.org/1qqq PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1qqq RCSB], [https://www.ebi.ac.uk/pdbsum/1qqq PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1qqq ProSAT]</span></td></tr>
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</table>
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== Function ==
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[https://www.uniprot.org/uniprot/TYSY_ECOLI TYSY_ECOLI] Provides the sole de novo source of dTMP for DNA biosynthesis. This protein also binds to its mRNA thus repressing its own translation.
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== Evolutionary Conservation ==
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[[Image:Consurf_key_small.gif|200px|right]]
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Check<jmol>
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<jmolCheckbox>
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<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/qq/1qqq_consurf.spt"</scriptWhenChecked>
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<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
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<text>to colour the structure by Evolutionary Conservation</text>
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</jmolCheckbox>
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</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=1qqq ConSurf].
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<div style="clear:both"></div>
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==About this Structure==
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==See Also==
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1QQQ 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 SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Thymidylate_synthase Thymidylate synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.45 2.1.1.45] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1QQQ OCA].
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*[[Thymidylate synthase 3D structures|Thymidylate synthase 3D structures]]
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__TOC__
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==Reference==
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</StructureSection>
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Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254., Fantz C, Shaw D, Jennings W, Forsthoefel A, Kitchens M, Phan J, Minor W, Lebioda L, Berger FG, Spencer HT, Mol Pharmacol. 2000 Feb;57(2):359-66. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10648646 10648646]
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[[Category: Escherichia coli]]
[[Category: Escherichia coli]]
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[[Category: Single protein]]
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[[Category: Large Structures]]
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[[Category: Thymidylate synthase]]
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[[Category: Berger FG]]
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[[Category: Berger, F.G.]]
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[[Category: Fantz C]]
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[[Category: Fantz, C.]]
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[[Category: Forsthoefel A]]
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[[Category: Forsthoefel, A.]]
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[[Category: Jennings W]]
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[[Category: Jennings, W.]]
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[[Category: Kitchens M]]
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[[Category: Kitchens, M.]]
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[[Category: Lebioda L]]
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[[Category: Lebioda, L.]]
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[[Category: Minor W]]
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[[Category: Minor, W.]]
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[[Category: Phan J]]
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[[Category: Phan, J.]]
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[[Category: Shaw D]]
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[[Category: Shaw, D.]]
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[[Category: Spencer HT]]
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[[Category: Spencer, H.T.]]
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[[Category: SO4]]
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[[Category: methyltransferase]]
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[[Category: thymidylate synthase]]
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[[Category: transferase]]
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''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 00:54:05 2007''
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Current revision

CRYSTAL STRUCTURE ANALYSIS OF SER254 MUTANT OF ESCHERICHIA COLI THYMIDYLATE SYNTHASE

PDB ID 1qqq

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