1cj2: Difference between revisions
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==MUTANT GLN34ARG OF PARA-HYDROXYBENZOATE HYDROXYLASE== | |||
<StructureSection load='1cj2' size='340' side='right'caption='[[1cj2]], [[Resolution|resolution]] 2.80Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[1cj2]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_fluorescens Pseudomonas fluorescens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CJ2 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1CJ2 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]] 2.8Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=FAD:FLAVIN-ADENINE+DINUCLEOTIDE'>FAD</scene>, <scene name='pdbligand=PHB:P-HYDROXYBENZOIC+ACID'>PHB</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=1cj2 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1cj2 OCA], [https://pdbe.org/1cj2 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1cj2 RCSB], [https://www.ebi.ac.uk/pdbsum/1cj2 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1cj2 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/PHHY_PSEFL PHHY_PSEFL] | |||
== 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/cj/1cj2_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=1cj2 ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase. | |||
Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.,Eppink MH, Overkamp KM, Schreuder HA, Van Berkel WJ J Mol Biol. 1999 Sep 10;292(1):87-96. PMID:10493859<ref>PMID:10493859</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 1cj2" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
[[ | *[[Hydroxylases 3D structures|Hydroxylases 3D structures]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
[[Category: | </StructureSection> | ||
[[Category: Large Structures]] | |||
[[Category: Pseudomonas fluorescens]] | [[Category: Pseudomonas fluorescens]] | ||
[[Category: Eppink MHM]] | |||
[[Category: Eppink | [[Category: Overkamp KM]] | ||
[[Category: Overkamp | [[Category: Schreuder HA]] | ||
[[Category: Schreuder | [[Category: Van Berkel WJH]] | ||
[[Category: | |||
Latest revision as of 08:52, 9 August 2023
MUTANT GLN34ARG OF PARA-HYDROXYBENZOATE HYDROXYLASEMUTANT GLN34ARG OF PARA-HYDROXYBENZOATE HYDROXYLASE
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 PubMedp-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase. Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.,Eppink MH, Overkamp KM, Schreuder HA, Van Berkel WJ J Mol Biol. 1999 Sep 10;292(1):87-96. PMID:10493859[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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