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[[Image:3drc.gif|left|200px]]<br /><applet load="3drc" size="350" color="white" frame="true" align="right" spinBox="true"
caption="3drc, resolution 1.9&Aring;" />
'''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS'''<br />


==Overview==
==INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS==
We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.
<StructureSection load='3drc' size='340' side='right'caption='[[3drc]], [[Resolution|resolution]] 1.90&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[3drc]] 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=3DRC OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3DRC 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]] 1.9&#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=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=MTX:METHOTREXATE'>MTX</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=3drc FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3drc OCA], [https://pdbe.org/3drc PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3drc RCSB], [https://www.ebi.ac.uk/pdbsum/3drc PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3drc ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/DYR_ECOLI DYR_ECOLI] Key enzyme in folate metabolism. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis.
== 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/dr/3drc_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=3drc ConSurf].
<div style="clear:both"></div>


==About this Structure==
==See Also==
3DRC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=CL:'>CL</scene>, <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=MTX:'>MTX</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Dihydrofolate_reductase Dihydrofolate reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.1.3 1.5.1.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3DRC OCA].
*[[Dihydrofolate reductase 3D structures|Dihydrofolate reductase 3D structures]]
 
__TOC__
==Reference==
</StructureSection>
Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis., Warren MS, Brown KA, Farnum MF, Howell EE, Kraut J, Biochemistry. 1991 Nov 19;30(46):11092-103. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=1932031 1932031]
[[Category: Dihydrofolate reductase]]
[[Category: Escherichia coli]]
[[Category: Escherichia coli]]
[[Category: Single protein]]
[[Category: Large Structures]]
[[Category: Kraut, J.]]
[[Category: Kraut J]]
[[Category: Oatley, S J.]]
[[Category: Oatley SJ]]
[[Category: CA]]
[[Category: CL]]
[[Category: MTX]]
[[Category: oxidoreductase]]
 
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 19:09:04 2008''

Latest revision as of 12:42, 21 February 2024

INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESISINVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS

Structural highlights

3drc is a 2 chain structure with sequence from Escherichia coli. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 1.9Å
Ligands:, ,
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

DYR_ECOLI Key enzyme in folate metabolism. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis.

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

See Also

3drc, resolution 1.90Å

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