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| [[Image:4cla.gif|left|200px]]<br /><applet load="4cla" size="350" color="white" frame="true" align="right" spinBox="true"
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| caption="4cla, resolution 2.0Å" />
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| '''ALTERNATIVE BINDING MODES FOR CHLORAMPHENICOL AND 1-SUBSTITUTED CHLORAMPHENICOL ANALOGUES REVEALED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY OF CHLORAMPHENICOL ACETYLTRANSFERASE'''<br />
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| ==Overview== | | ==ALTERNATIVE BINDING MODES FOR CHLORAMPHENICOL AND 1-SUBSTITUTED CHLORAMPHENICOL ANALOGUES REVEALED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY OF CHLORAMPHENICOL ACETYLTRANSFERASE== |
| Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol. The consequences of the substitution of Leu-160 by glutamine and by phenylalanine were deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-CoA to the 3-hydroxyl of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol. The acetyl group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar position to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160. In the case of Gln-160 CAT, large increases in Km for the three acetyl acceptors were accompanied by small decreases in kcat and in apparent affinity for acetyl-CoA. Such results are consistent with the introduction of the relatively hydrophilic amide in place of the delta-methyl groups of Leu-160. The kinetic properties of Phe-160 CAT were unexpected in that Km for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas kcat fell significantly (44-83-fold) in each case. The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with the alternative acyl acceptors were similar for wild-type and mutant enzymes. As the residue substitutions for Leu-160 do not result in enhanced discrimination against the binding and acetylation of 1-acetylchloramphenicol, it appears unlikely that the 1-acetyl group binds to the CAT active site in the same position as that occupied by the 1-hydroxyl of chloramphenicol.(ABSTRACT TRUNCATED AT 250 WORDS)
| | <StructureSection load='4cla' size='340' side='right'caption='[[4cla]], [[Resolution|resolution]] 2.00Å' scene=''> |
| | == Structural highlights == |
| | <table><tr><td colspan='2'>[[4cla]] 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=4CLA OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=4CLA 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]] 2Å</td></tr> |
| | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CLM:CHLORAMPHENICOL'>CLM</scene>, <scene name='pdbligand=CO:COBALT+(II)+ION'>CO</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=4cla FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4cla OCA], [https://pdbe.org/4cla PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=4cla RCSB], [https://www.ebi.ac.uk/pdbsum/4cla PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=4cla ProSAT]</span></td></tr> |
| | </table> |
| | == Function == |
| | [https://www.uniprot.org/uniprot/CAT3_ECOLX CAT3_ECOLX] This enzyme is an effector of chloramphenicol resistance in bacteria. |
| | == 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/cl/4cla_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=4cla ConSurf]. |
| | <div style="clear:both"></div> |
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| ==About this Structure== | | ==See Also== |
| 4CLA 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=CO:'>CO</scene> and <scene name='pdbligand=CLM:'>CLM</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Chloramphenicol_O-acetyltransferase Chloramphenicol O-acetyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.28 2.3.1.28] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4CLA OCA].
| | *[[Chloramphenicol acetyltransferase 3D structures|Chloramphenicol acetyltransferase 3D structures]] |
| | | __TOC__ |
| ==Reference==
| | </StructureSection> |
| Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase., Murray IA, Lewendon A, Williams JA, Cullis PM, Shaw WV, Leslie AG, Biochemistry. 1991 Apr 16;30(15):3763-70. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=2015231 2015231]
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| [[Category: Chloramphenicol O-acetyltransferase]]
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| [[Category: Escherichia coli]] | | [[Category: Escherichia coli]] |
| [[Category: Single protein]] | | [[Category: Large Structures]] |
| [[Category: Leslie, A G.W.]] | | [[Category: Leslie AGW]] |
| [[Category: CLM]]
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| [[Category: CO]]
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| [[Category: transferase (acyltransferase)]]
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| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 19:12:56 2008''
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