3fyp: Difference between revisions
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[[Image: | ==Crystal structure of the quadruple mutant (N23C/C246S/D247E/P249A) of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) from Neisseria meningitidis== | ||
<StructureSection load='3fyp' size='340' side='right' caption='[[3fyp]], [[Resolution|resolution]] 1.85Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[3fyp]] is a 4 chain structure with sequence from [http://en.wikipedia.org/wiki/Neisseria_meningitidis_serogroup_b Neisseria meningitidis serogroup b]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3FYP OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3FYP FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=GOL:GLYCEROL'>GOL</scene>, <scene name='pdbligand=MN:MANGANESE+(II)+ION'>MN</scene>, <scene name='pdbligand=NA:SODIUM+ION'>NA</scene>, <scene name='pdbligand=PEP:PHOSPHOENOLPYRUVATE'>PEP</scene></td></tr> | |||
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[2qkf|2qkf]]</td></tr> | |||
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">kdsA, NMB1283 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=491 Neisseria meningitidis serogroup B])</td></tr> | |||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/3-deoxy-8-phosphooctulonate_synthase 3-deoxy-8-phosphooctulonate synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.5.1.55 2.5.1.55] </span></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3fyp FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3fyp OCA], [http://www.rcsb.org/pdb/explore.do?structureId=3fyp RCSB], [http://www.ebi.ac.uk/pdbsum/3fyp PDBsum]</span></td></tr> | |||
</table> | |||
== 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/fy/3fyp_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/chain_selection.php?pdb_ID=2ata ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Two distinct groups of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved (246)CysAspGlyPro(249) motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd(2+), the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 A. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu(2+). Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs. | |||
Reversing evolution: re-establishing obligate metal ion dependence in a metal-independent KDO8P synthase.,Cochrane FC, Cookson TV, Jameson GB, Parker EJ J Mol Biol. 2009 Jul 24;390(4):646-61. Epub 2009 May 15. PMID:19447118<ref>PMID:19447118</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | ==See Also== | ||
*[[Kdo-8-phosphate synthase|Kdo-8-phosphate synthase]] | *[[Kdo-8-phosphate synthase|Kdo-8-phosphate synthase]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: 3-deoxy-8-phosphooctulonate synthase]] | [[Category: 3-deoxy-8-phosphooctulonate synthase]] | ||
[[Category: Neisseria meningitidis serogroup b]] | [[Category: Neisseria meningitidis serogroup b]] | ||
[[Category: Cochrane, F P | [[Category: Cochrane, F P]] | ||
[[Category: Jameson, G B | [[Category: Jameson, G B]] | ||
[[Category: Parker, E J | [[Category: Parker, E J]] | ||
[[Category: Patchett, M L | [[Category: Patchett, M L]] | ||
[[Category: Biosynthesis]] | [[Category: Biosynthesis]] | ||
[[Category: Kdo8 kdop]] | [[Category: Kdo8 kdop]] | ||
Revision as of 13:18, 3 December 2014
Crystal structure of the quadruple mutant (N23C/C246S/D247E/P249A) of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) from Neisseria meningitidisCrystal structure of the quadruple mutant (N23C/C246S/D247E/P249A) of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) from Neisseria meningitidis
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedTwo distinct groups of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved (246)CysAspGlyPro(249) motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd(2+), the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 A. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu(2+). Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs. Reversing evolution: re-establishing obligate metal ion dependence in a metal-independent KDO8P synthase.,Cochrane FC, Cookson TV, Jameson GB, Parker EJ J Mol Biol. 2009 Jul 24;390(4):646-61. Epub 2009 May 15. PMID:19447118[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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