3h4h: Difference between revisions
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< | ==Met94Thr/Phe312Cys variant of nitrite reductase from Alcaligenes faecalis== | ||
<StructureSection load='3h4h' size='340' side='right'caption='[[3h4h]], [[Resolution|resolution]] 1.60Å' scene=''> | |||
You may | == Structural highlights == | ||
<table><tr><td colspan='2'>[[3h4h]] 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=3H4H OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3H4H 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]] 1.6Å</td></tr> | |||
-- | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CSD:3-SULFINOALANINE'>CSD</scene>, <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=3h4h FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3h4h OCA], [https://pdbe.org/3h4h PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3h4h RCSB], [https://www.ebi.ac.uk/pdbsum/3h4h PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3h4h 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/h4/3h4h_consurf.spt"</scriptWhenChecked> | |||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview03.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=3h4h 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 3h4h" style="background-color:#fffaf0;"></div> | |||
==See Also== | |||
*[[Nitrite reductase 3D structures|Nitrite reductase 3D structures]] | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
== | |||
== | |||
< | |||
[[Category: Alcaligenes faecalis]] | [[Category: Alcaligenes faecalis]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: | [[Category: MacPherson IS]] | ||
[[Category: | [[Category: Murphy IS]] | ||
Latest revision as of 12:13, 30 October 2024
Met94Thr/Phe312Cys variant of nitrite reductase from Alcaligenes faecalisMet94Thr/Phe312Cys variant of nitrite reductase from Alcaligenes faecalis
Structural highlights
FunctionEvolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedDirected 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[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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