3h4f: Difference between revisions
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New page: '''Unreleased structure''' The entry 3h4f is ON HOLD Authors: MacPherson, I.S., Rosell, F.I., Scofield, M., Mauk, A.G., Murphy, M.E.P. Description: Met62Leu variant of nitrite reductas... |
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The | ==Met62Leu variant of nitrite reductase from Alcaligenes faeclis== | ||
<StructureSection load='3h4f' size='340' side='right'caption='[[3h4f]], [[Resolution|resolution]] 2.10Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[3h4f]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Alcaligenes_faecalis Alcaligenes faecalis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3H4F OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3H4F 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]] 2.1Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CU:COPPER+(II)+ION'>CU</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=3h4f FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3h4f OCA], [https://pdbe.org/3h4f PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3h4f RCSB], [https://www.ebi.ac.uk/pdbsum/3h4f PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3h4f ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/NIR_ALCFA NIR_ALCFA] | |||
== 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/h4/3h4f_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=3h4f ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs. | |||
Directed evolution of copper nitrite reductase to a chromogenic reductant.,MacPherson IS, Rosell FI, Scofield M, Mauk AG, Murphy ME Protein Eng Des Sel. 2010 Mar;23(3):137-45. Epub 2010 Jan 18. PMID:20083495<ref>PMID:20083495</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 3h4f" style="background-color:#fffaf0;"></div> | |||
==See Also== | |||
*[[Nitrite reductase 3D structures|Nitrite reductase 3D structures]] | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Alcaligenes faecalis]] | |||
[[Category: Large Structures]] | |||
[[Category: MacPherson IS]] | |||
[[Category: Mauk AG]] | |||
[[Category: Murphy MEP]] | |||
[[Category: Rosell FI]] | |||
[[Category: Scofield M]] | |||
Latest revision as of 07:15, 6 September 2023
Met62Leu variant of nitrite reductase from Alcaligenes faeclis
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