1ev8

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(New page: 200px<br /><applet load="1ev8" size="450" color="white" frame="true" align="right" spinBox="true" caption="1ev8, resolution 2.60&Aring;" /> '''CRYSTAL STRUCTURE AN...)
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[[Image:1ev8.jpg|left|200px]]<br /><applet load="1ev8" size="450" color="white" frame="true" align="right" spinBox="true"
 
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caption="1ev8, resolution 2.60&Aring;" />
 
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'''CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI'''<br />
 
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==Overview==
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==CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI==
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The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in, catalysis has been characterized by kinetic and crystallographic studies., Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of, E. coli in medium lacking thymidine. Steady-state kinetic analysis, revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower, than that of the wild-type enzyme. Relative to wild-type TS, K(m) values, of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate), were 1.5-16-fold higher. The rate of dehalogenation of, 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by, TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was, analyzed and showed that only S167A and S167G catalyzed the dehalogenation, reaction and values of k(cat)/K(m) for the mutant enzymes were decreased, by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics, of ternary complex formation revealed that the productive binding of, CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme., Chemical transformation constants (k(chem)) for the mutant enzymes were, lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and, S167C crystallized in the I2(1)3 space group and scattered X-rays to, either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data, sets were refined to a R(crys) of 19.9%. In the crystals some cysteine, residues were derivatized with 2-mercaptoethanol to form, S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates, that in the absence of bound substrate the catalytic cysteine is not more, reactive than other cysteines. It is proposed that the catalytic cysteine, is activated by substrate binding by a proton-transfer mechanism in which, the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His, 207-Asp 205 diad via a system of ordered water molecules.
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<StructureSection load='1ev8' size='340' side='right'caption='[[1ev8]], [[Resolution|resolution]] 2.60&Aring;' scene=''>
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== Structural highlights ==
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<table><tr><td colspan='2'>[[1ev8]] 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=1EV8 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1EV8 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]] 2.6&#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></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=1ev8 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ev8 OCA], [https://pdbe.org/1ev8 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1ev8 RCSB], [https://www.ebi.ac.uk/pdbsum/1ev8 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1ev8 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/ev/1ev8_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=1ev8 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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1EV8 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. 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=1EV8 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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Catalytic cysteine of thymidylate synthase is activated upon substrate binding., Phan J, Mahdavian E, Nivens MC, Minor W, Berger S, Spencer HT, Dunlap RB, Lebioda L, Biochemistry. 2000 Jun 13;39(23):6969-78. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10841779 10841779]
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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 S]]
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[[Category: Berger, S.]]
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[[Category: Dunlap RB]]
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[[Category: Dunlap, R.B.]]
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[[Category: Lebioda L]]
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[[Category: Lebioda, L.]]
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[[Category: Mahdavian E]]
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[[Category: Mahdavian, E.]]
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[[Category: Minor W]]
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[[Category: Minor, W.]]
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[[Category: Nivens MC]]
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[[Category: Nivens, M.C.]]
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[[Category: Phan J]]
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[[Category: Phan, J.]]
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[[Category: Spencer HT]]
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[[Category: Spencer, H.T.]]
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[[Category: cys167 e. coli thymidylate synthase]]
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''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 14:20:47 2007''
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CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI

PDB ID 1ev8

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