6grr: Difference between revisions
New page: '''Unreleased structure''' The entry 6grr is ON HOLD Authors: Gaule, T.G., Smith, M.A., Tych, K.M., Pirrat, P., Trinh, C.H., Pearson, A.R., Knowles, P.F., McPherson, M.J. Description: ... |
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The | ==Crystal structure of Escherichia coli amine oxidase mutant I342F/E573Q== | ||
<StructureSection load='6grr' size='340' side='right' caption='[[6grr]], [[Resolution|resolution]] 1.70Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[6grr]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/"bacillus_coli"_migula_1895 "bacillus coli" migula 1895]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6GRR OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6GRR FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=CU:COPPER+(II)+ION'>CU</scene>, <scene name='pdbligand=GOL:GLYCEROL'>GOL</scene></td></tr> | |||
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">RK56_010715 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=562 "Bacillus coli" Migula 1895])</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=6grr FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6grr OCA], [http://pdbe.org/6grr PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6grr RCSB], [http://www.ebi.ac.uk/pdbsum/6grr PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6grr ProSAT]</span></td></tr> | |||
</table> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu(2+) in the active site, two peripheral calcium sites, approximately 32 A from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca(2+) (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca(2+) (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis ( approximately 60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity ( approximately 4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu(2+)-substrate reduced TPQ (TPQAMQ) Cu(+)-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu(2+)-TPQAMQ or destabilizes Cu(+)-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu(2+)-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu(+)-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis. | |||
Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli.,Gaule TG, Smith MA, Tych KM, Pirrat P, Trinh CH, Pearson AR, Knowles PF, McPherson MJ Biochemistry. 2018 Aug 24. doi: 10.1021/acs.biochem.8b00633. PMID:30110143<ref>PMID:30110143</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: | <div class="pdbe-citations 6grr" style="background-color:#fffaf0;"></div> | ||
[[Category: | == References == | ||
[[Category: | <references/> | ||
[[Category: | __TOC__ | ||
</StructureSection> | |||
[[Category: Bacillus coli migula 1895]] | |||
[[Category: Gaule, T G]] | |||
[[Category: Knowles, P F]] | |||
[[Category: McPherson, M J]] | |||
[[Category: Pearson, A R]] | |||
[[Category: Pirrat, P]] | [[Category: Pirrat, P]] | ||
[[Category: | [[Category: Smith, M A]] | ||
[[Category: Trinh, C | [[Category: Trinh, C H]] | ||
[[Category: Tych, K M]] | |||
[[Category: Oxidoreductase]] | |||
Latest revision as of 23:23, 19 September 2018
Crystal structure of Escherichia coli amine oxidase mutant I342F/E573QCrystal structure of Escherichia coli amine oxidase mutant I342F/E573Q
Structural highlights
Publication Abstract from PubMedCopper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu(2+) in the active site, two peripheral calcium sites, approximately 32 A from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca(2+) (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca(2+) (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis ( approximately 60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity ( approximately 4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu(2+)-substrate reduced TPQ (TPQAMQ) Cu(+)-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu(2+)-TPQAMQ or destabilizes Cu(+)-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu(2+)-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu(+)-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis. Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli.,Gaule TG, Smith MA, Tych KM, Pirrat P, Trinh CH, Pearson AR, Knowles PF, McPherson MJ Biochemistry. 2018 Aug 24. doi: 10.1021/acs.biochem.8b00633. PMID:30110143[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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