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[[Image:1w1k.gif|left|200px]]<br />
<applet load="1w1k" size="450" color="white" frame="true" align="right" spinBox="true"
caption="1w1k, resolution 2.55&Aring;" />
'''STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: ILE238THR MUTANT'''<br />


==Overview==
==STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: Ile238Thr Mutant==
The flavoenzyme vanillyl-alcohol oxidase was subjected to random, mutagenesis to generate mutants with enhanced reactivity to creosol, (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated, conversion of creosol proceeds via a two-step process in which the, initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is, oxidized to the widely used flavor compound vanillin, (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is, extremely slow due to the formation of a covalent FAD N-5-creosol adduct., After a single round of error-prone PCR, seven mutants were generated with, increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency, (kcat/Km) with creosol compared with the wild-type enzyme. This enhanced, reactivity was due to a lower stability of the covalent flavin-substrate, adduct, thereby promoting vanillin formation. The catalytic efficiencies, of the mutants were also enhanced for other ortho-substituted, 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino, acid residues are not located within a distance of direct interaction with, the substrate, and the determined three-dimensional structures of the, mutant enzymes are highly similar to that of the wild-type enzyme. These, results clearly show the importance of remote residues, not readily, predicted by rational design, for the substrate specificity of enzymes.
<StructureSection load='1w1k' size='340' side='right'caption='[[1w1k]], [[Resolution|resolution]] 2.55&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[1w1k]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Penicillium_simplicissimum Penicillium simplicissimum]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1W1K OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1W1K 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]] 2.55&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=EUG:2-METHOXY-4-[(1E)-PROP-1-EN-1-YL]PHENOL'>EUG</scene>, <scene name='pdbligand=FAD:FLAVIN-ADENINE+DINUCLEOTIDE'>FAD</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=1w1k FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1w1k OCA], [https://pdbe.org/1w1k PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1w1k RCSB], [https://www.ebi.ac.uk/pdbsum/1w1k PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1w1k ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/VAOX_PENSI VAOX_PENSI] Catalyzes the conversion of vanillin alcohol to vanillin, and also the conversion of a wide range of phenolic compounds bearing side chains of variable size at the para position of the aromatic ring. Crucial for the degradation of the secondary metabolites derived from the degradation of the lignin. Catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines, the oxidative demethylation of 4-(methoxy-methyl)phenols and the oxidative hydration of 4-allylphenols. Most active with 4-allylphenols.
== 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/w1/1w1k_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=1w1k ConSurf].
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (kcat/Km) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.


==About this Structure==
Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin.,van den Heuvel RH, van den Berg WA, Rovida S, van Berkel WJ J Biol Chem. 2004 Aug 6;279(32):33492-500. Epub 2004 May 28. PMID:15169773<ref>PMID:15169773</ref>
1W1K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Penicillium_simplicissimum Penicillium simplicissimum] with FAD and EUG as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alcohol_oxidase Alcohol oxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.13 1.1.3.13] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1W1K OCA].


==Reference==
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin., van den Heuvel RH, van den Berg WA, Rovida S, van Berkel WJ, J Biol Chem. 2004 Aug 6;279(32):33492-500. Epub 2004 May 28. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=15169773 15169773]
</div>
[[Category: Alcohol oxidase]]
<div class="pdbe-citations 1w1k" style="background-color:#fffaf0;"></div>
 
==See Also==
*[[Vanillyl-alcohol oxidase|Vanillyl-alcohol oxidase]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Penicillium simplicissimum]]
[[Category: Penicillium simplicissimum]]
[[Category: Single protein]]
[[Category: Van Den Heuvel RH]]
[[Category: Heuvel, R.H.Van.Den.]]
[[Category: EUG]]
[[Category: FAD]]
[[Category: catalysis]]
[[Category: fad]]
[[Category: flavoenzyme]]
[[Category: oxidoreductase]]
 
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov  5 14:20:25 2007''

Latest revision as of 03:37, 21 November 2024

STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: Ile238Thr MutantSTRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: Ile238Thr Mutant

Structural highlights

1w1k is a 2 chain structure with sequence from Penicillium simplicissimum. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 2.55Å
Ligands:,
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

VAOX_PENSI Catalyzes the conversion of vanillin alcohol to vanillin, and also the conversion of a wide range of phenolic compounds bearing side chains of variable size at the para position of the aromatic ring. Crucial for the degradation of the secondary metabolites derived from the degradation of the lignin. Catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines, the oxidative demethylation of 4-(methoxy-methyl)phenols and the oxidative hydration of 4-allylphenols. Most active with 4-allylphenols.

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 PubMed

The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (kcat/Km) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.

Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin.,van den Heuvel RH, van den Berg WA, Rovida S, van Berkel WJ J Biol Chem. 2004 Aug 6;279(32):33492-500. Epub 2004 May 28. PMID:15169773[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

See Also

References

  1. ↑ van den Heuvel RH, van den Berg WA, Rovida S, van Berkel WJ. Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin. J Biol Chem. 2004 Aug 6;279(32):33492-500. Epub 2004 May 28. PMID:15169773 doi:10.1074/jbc.M312968200

1w1k, resolution 2.55Å

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