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| [[Image:1a5k.gif|left|200px]]<br /><applet load="1a5k" size="350" color="white" frame="true" align="right" spinBox="true"
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| caption="1a5k, resolution 2.2Å" />
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| '''K217E VARIANT OF KLEBSIELLA AEROGENES UREASE'''<br />
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| ==Overview== | | ==K217E VARIANT OF KLEBSIELLA AEROGENES UREASE== |
| Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis. | | <StructureSection load='1a5k' size='340' side='right'caption='[[1a5k]], [[Resolution|resolution]] 2.20Å' scene=''> |
| | == Structural highlights == |
| | <table><tr><td colspan='2'>[[1a5k]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Klebsiella_aerogenes Klebsiella aerogenes]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1A5K OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1A5K 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]] 2.2Å</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=1a5k FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1a5k OCA], [https://pdbe.org/1a5k PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1a5k RCSB], [https://www.ebi.ac.uk/pdbsum/1a5k PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1a5k ProSAT]</span></td></tr> |
| | </table> |
| | == Function == |
| | [https://www.uniprot.org/uniprot/URE3_KLEAE URE3_KLEAE] |
| | == 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/a5/1a5k_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=1a5k ConSurf]. |
| | <div style="clear:both"></div> |
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| ==About this Structure== | | ==See Also== |
| 1A5K is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Klebsiella_aerogenes Klebsiella aerogenes]. Active as [http://en.wikipedia.org/wiki/Urease Urease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.5 3.5.1.5] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1A5K OCA].
| | *[[Urease 3D structures|Urease 3D structures]] |
| | | __TOC__ |
| ==Reference==
| | </StructureSection> |
| Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand., Pearson MA, Schaller RA, Michel LO, Karplus PA, Hausinger RP, Biochemistry. 1998 Apr 28;37(17):6214-20. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=9558361 9558361]
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| [[Category: Klebsiella aerogenes]] | | [[Category: Klebsiella aerogenes]] |
| [[Category: Protein complex]] | | [[Category: Large Structures]] |
| [[Category: Urease]]
| | [[Category: Hausinger RP]] |
| [[Category: Hausinger, R P.]] | | [[Category: Karplus PA]] |
| [[Category: Karplus, P A.]] | | [[Category: Michel LO]] |
| [[Category: Michel, L O.]] | | [[Category: Pearson MA]] |
| [[Category: Pearson, M A.]] | | [[Category: Schaller RA]] |
| [[Category: Schaller, R A.]] | |
| [[Category: hydrolase (urea amido)]]
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| [[Category: mutant]]
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| [[Category: nickel metalloenzyme]]
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| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:41:05 2008''
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