3ir7: Difference between revisions
No edit summary |
No edit summary |
||
| (9 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
< | ==Crystal structure of NarGHI mutant NarG-R94S== | ||
<StructureSection load='3ir7' size='340' side='right'caption='[[3ir7]], [[Resolution|resolution]] 2.50Å' scene=''> | |||
You may | == Structural highlights == | ||
<table><tr><td colspan='2'>[[3ir7]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli_K-12 Escherichia coli K-12]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3IR7 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3IR7 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]] 2.5Å</td></tr> | |||
-- | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=6MO:MOLYBDENUM(VI)+ION'>6MO</scene>, <scene name='pdbligand=AGA:(1S)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PENTANOYLOXY)METHYL]ETHYL+OCTANOATE'>AGA</scene>, <scene name='pdbligand=FME:N-FORMYLMETHIONINE'>FME</scene>, <scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene>, <scene name='pdbligand=MD1:PHOSPHORIC+ACID+4-(2-AMINO-4-OXO-3,4,5,6,-TETRAHYDRO-PTERIDIN-6-YL)-2-HYDROXY-3,4-DIMERCAPTO-BUT-3-EN-YL+ESTER+GUANYLATE+ESTER'>MD1</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=3ir7 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3ir7 OCA], [https://pdbe.org/3ir7 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3ir7 RCSB], [https://www.ebi.ac.uk/pdbsum/3ir7 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3ir7 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/NARG_ECOLI NARG_ECOLI] The nitrate reductase enzyme complex allows E.coli to use nitrate as an electron acceptor during anaerobic growth. The alpha chain is the actual site of nitrate reduction. | |||
== 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/ir/3ir7_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=3ir7 ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His(49) both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E(m) value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the Mo-bisPGD cofactor. To demonstrate that decreasing the FS0 cluster E(m) results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg(94)) in the vicinity of FS0 to a Ser residue. In this case, the E(m) of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to approximately 30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, (49)HGVNCTG(55), must be correctly positioned to ensure holoenzyme maturation. | |||
Protein crystallography reveals a role for the FS0 cluster of Escherichia coli nitrate reductase A (NarGHI) in enzyme maturation.,Rothery RA, Bertero MG, Spreter T, Bouromand N, Strynadka NC, Weiner JH J Biol Chem. 2010 Mar 19;285(12):8801-7. Epub 2010 Jan 6. PMID:20053990<ref>PMID:20053990</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 3ir7" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
*[[Nitrate reductase|Nitrate reductase]] | |||
[[Category: Escherichia coli | == References == | ||
[[Category: | <references/> | ||
[[Category: Bertero | __TOC__ | ||
[[Category: Rothery | </StructureSection> | ||
[[Category: Strynadka | [[Category: Escherichia coli K-12]] | ||
[[Category: Weiner | [[Category: Large Structures]] | ||
[[Category: Bertero MG]] | |||
[[Category: Rothery RA]] | |||
[[Category: Strynadka NCJ]] | |||
[[Category: Weiner JH]] | |||
Latest revision as of 12:18, 30 October 2024
Crystal structure of NarGHI mutant NarG-R94SCrystal structure of NarGHI mutant NarG-R94S
Structural highlights
FunctionNARG_ECOLI The nitrate reductase enzyme complex allows E.coli to use nitrate as an electron acceptor during anaerobic growth. The alpha chain is the actual site of nitrate reduction. 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 PubMedWe have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His(49) both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E(m) value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the Mo-bisPGD cofactor. To demonstrate that decreasing the FS0 cluster E(m) results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg(94)) in the vicinity of FS0 to a Ser residue. In this case, the E(m) of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to approximately 30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, (49)HGVNCTG(55), must be correctly positioned to ensure holoenzyme maturation. Protein crystallography reveals a role for the FS0 cluster of Escherichia coli nitrate reductase A (NarGHI) in enzyme maturation.,Rothery RA, Bertero MG, Spreter T, Bouromand N, Strynadka NC, Weiner JH J Biol Chem. 2010 Mar 19;285(12):8801-7. Epub 2010 Jan 6. PMID:20053990[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
|
| ||||||||||||||||||