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==Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing== | |||
<StructureSection load='1ghd' size='340' side='right'caption='[[1ghd]], [[Resolution|resolution]] 2.40Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[1ghd]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_sp._130 Pseudomonas sp. 130]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1GHD OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1GHD 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]] 2.4Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=MSE:SELENOMETHIONINE'>MSE</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=1ghd FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ghd OCA], [https://pdbe.org/1ghd PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1ghd RCSB], [https://www.ebi.ac.uk/pdbsum/1ghd PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1ghd ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/O86089_9PROT O86089_9PROT] | |||
== 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/gh/1ghd_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=1ghd ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis. | |||
Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.,Huang X, Zeng R, Ding X, Mao X, Ding Y, Rao Z, Xie Y, Jiang W, Zhao G J Biol Chem. 2002 Mar 22;277(12):10256-64. Epub 2002 Jan 8. PMID:11782466<ref>PMID:11782466</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 1ghd" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
*[[Cephalosporin acylase 3D structures|Cephalosporin acylase 3D structures]] | |||
[[Category: | == References == | ||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Pseudomonas sp. 130]] | [[Category: Pseudomonas sp. 130]] | ||
[[Category: Bartlam | [[Category: Bartlam M]] | ||
[[Category: Ding | [[Category: Ding Y]] | ||
[[Category: He | [[Category: He H]] | ||
[[Category: Jiang | [[Category: Jiang F]] | ||
[[Category: Jiang | [[Category: Jiang W]] | ||
[[Category: Liu | [[Category: Liu Y]] | ||
[[Category: Mao | [[Category: Mao X]] | ||
[[Category: Rao | [[Category: Rao Z]] | ||
[[Category: Tang | [[Category: Tang H]] | ||
[[Category: Ye | [[Category: Ye S]] | ||
[[Category: Zhang | [[Category: Zhang S]] | ||
[[Category: Zhao | [[Category: Zhao G]] | ||
Latest revision as of 11:27, 6 November 2024
Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasingCrystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing
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 PubMedGlutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis. Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.,Huang X, Zeng R, Ding X, Mao X, Ding Y, Rao Z, Xie Y, Jiang W, Zhao G J Biol Chem. 2002 Mar 22;277(12):10256-64. Epub 2002 Jan 8. PMID:11782466[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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