1h2g: Difference between revisions

From Proteopedia
Jump to navigationJump to search
← Older edit
OCA (talk | contribs)
Bots, Bureaucrats, journal, Administrators
3,331,097 edits
No edit summary
OCA (talk | contribs)
Bots, Bureaucrats, journal, Administrators
3,331,097 edits
No edit summary
 
(20 intermediate revisions by the same user not shown)
Line 1: Line 1:
[[Image:1h2g.gif|left|200px]]<br />
<applet load="1h2g" size="450" color="white" frame="true" align="right" spinBox="true"
caption="1h2g, resolution 2.00&Aring;" />
'''ALTERED SUBSTRATE SPECIFICITY MUTANT OF PENICILLIN ACYLASE'''<br />


==Overview==
==Altered substrate specificity mutant of penicillin acylase==
Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use, glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl., Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases, of up to a factor of ten in k (cat)/ K (m) values for substrates, possessing a phenylacetyl leaving group are consistent with a decrease in, K (s). Values of k (cat)/ K (m) for glutaryl-L-leucine are increased at, least 100-fold. A decrease in k (cat)/ K (m) for the Cys(B71) mutant with, increased pH is consistent with binding of the uncharged glutaryl group., The mutant proteins are more resistant to urea denaturation monitored by, protein fluorescence, to inactivation in the presence of substrate either, in the presence of urea or at high pH, and to heat inactivation. The, crystal structure of the Leu(B71) mutant protein, solved to 2 A, resolution, shows a flip of the side chain of Phe(B256) into the periphery, of the catalytic centre, associated with loss of the pi-stacking, interactions between Phe(B256) and Phe(B71). Molecular modelling, demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl, group in the S(1) subsite of either the wild-type or the Leu(B71) mutant, but with greater potential freedom of rotation of the substrate leucine, moiety in the complex with the mutant protein. This implies a smaller, decrease in the conformational entropy of the substrate on binding to the, mutant proteins and consequently greater catalytic activity.
<StructureSection load='1h2g' size='340' side='right'caption='[[1h2g]], [[Resolution|resolution]] 2.00&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[1h2g]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1H2G OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1H2G 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&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</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=1h2g FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1h2g OCA], [https://pdbe.org/1h2g PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1h2g RCSB], [https://www.ebi.ac.uk/pdbsum/1h2g PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1h2g ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/PAC_ECOLX PAC_ECOLX]
== 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/h2/1h2g_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=1h2g ConSurf].
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k (cat)/ K (m) values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K (s). Values of k (cat)/ K (m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k (cat)/ K (m) for the Cys(B71) mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 A resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S(1) subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity.


==About this Structure==
Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding.,Morillas M, McVey CE, Brannigan JA, Ladurner AG, Forney LJ, Virden R Biochem J. 2003 Apr 1;371(Pt 1):143-50. PMID:12511194<ref>PMID:12511194</ref>
1H2G is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CA and EDO as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Penicillin_amidase Penicillin amidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.11 3.5.1.11] Structure known Active Site: CA1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1H2G OCA].


==Reference==
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding., Morillas M, McVey CE, Brannigan JA, Ladurner AG, Forney LJ, Virden R, Biochem J. 2003 Apr 1;371(Pt 1):143-50. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=12511194 12511194]
</div>
<div class="pdbe-citations 1h2g" style="background-color:#fffaf0;"></div>
 
==See Also==
*[[Penicillin acylase|Penicillin acylase]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Escherichia coli]]
[[Category: Escherichia coli]]
[[Category: Penicillin amidase]]
[[Category: Large Structures]]
[[Category: Protein complex]]
[[Category: Brannigan JA]]
[[Category: Brannigan, J.A.]]
[[Category: Forney LJ]]
[[Category: Forney, L.J.]]
[[Category: Ladurner AG]]
[[Category: Ladurner, A.G.]]
[[Category: McVey CE]]
[[Category: Mcvey, C.E.]]
[[Category: Morillas M]]
[[Category: Morillas, M.]]
[[Category: Virden R]]
[[Category: Virden, R.]]
[[Category: CA]]
[[Category: EDO]]
[[Category: altered specificity]]
[[Category: amidohydrolase]]
[[Category: antibiotic resistance]]
[[Category: hydrolase]]
[[Category: zymogen]]
 
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov  5 13:57:10 2007''
Retrieved from "https://proteopedia.org/1h2g"