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==METHOTREXATE-RESISTANT VARIANTS OF HUMAN DIHYDROFOLATE REDUCTASE WITH SUBSTITUTION OF LEUCINE 22: KINETICS, CRYSTALLOGRAPHY AND POTENTIAL AS SELECTABLE MARKERS==
==METHOTREXATE-RESISTANT VARIANTS OF HUMAN DIHYDROFOLATE REDUCTASE WITH SUBSTITUTION OF LEUCINE 22: KINETICS, CRYSTALLOGRAPHY AND POTENTIAL AS SELECTABLE MARKERS==
<StructureSection load='1dls' size='340' side='right' caption='[[1dls]], [[Resolution|resolution]] 2.30&Aring;' scene=''>
<StructureSection load='1dls' size='340' side='right'caption='[[1dls]], [[Resolution|resolution]] 2.30&Aring;' scene=''>
== Structural highlights ==
== Structural highlights ==
<table><tr><td colspan='2'>[[1dls]] is a 1 chain structure with sequence from [http://en.wikipedia.org/wiki/Human Human]. The October 2002 RCSB PDB [http://pdb.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/index.html Molecule of the Month] feature on ''Dihydrofolate Reductase''  by David S. Goodsell is [http://dx.doi.org/10.2210/rcsb_pdb/mom_2002_10 10.2210/rcsb_pdb/mom_2002_10]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1DLS OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1DLS FirstGlance]. <br>
<table><tr><td colspan='2'>[[1dls]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. The October 2002 RCSB PDB [https://pdb.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/index.html Molecule of the Month] feature on ''Dihydrofolate Reductase''  by David S. Goodsell is [https://dx.doi.org/10.2210/rcsb_pdb/mom_2002_10 10.2210/rcsb_pdb/mom_2002_10]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1DLS OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1DLS FirstGlance]. <br>
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=MTX:METHOTREXATE'>MTX</scene>, <scene name='pdbligand=NDP:NADPH+DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE+PHOSPHATE'>NDP</scene></td></tr>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.3&#8491;</td></tr>
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">POTENTIAL ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=9606 HUMAN])</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=MTX:METHOTREXATE'>MTX</scene>, <scene name='pdbligand=NDP:NADPH+DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE+PHOSPHATE'>NDP</scene></td></tr>
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[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] </span></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=1dls FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1dls OCA], [https://pdbe.org/1dls PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1dls RCSB], [https://www.ebi.ac.uk/pdbsum/1dls PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1dls ProSAT]</span></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1dls FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1dls OCA], [http://pdbe.org/1dls PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=1dls RCSB], [http://www.ebi.ac.uk/pdbsum/1dls PDBsum]</span></td></tr>
</table>
</table>
== Disease ==
== Disease ==
[[http://www.uniprot.org/uniprot/DYR_HUMAN DYR_HUMAN]] Defects in DHFR are the cause of megaloblastic anemia due to dihydrofolate reductase deficiency (DHFRD) [MIM:[http://omim.org/entry/613839 613839]]. DHFRD is an inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency. Clinical features include variable neurologic symptoms, ranging from severe developmental delay and generalized seizures in infancy, to childhood absence epilepsy with learning difficulties, to lack of symptoms.<ref>PMID:21310276</ref> <ref>PMID:21310277</ref>
[https://www.uniprot.org/uniprot/DYR_HUMAN DYR_HUMAN] Defects in DHFR are the cause of megaloblastic anemia due to dihydrofolate reductase deficiency (DHFRD) [MIM:[https://omim.org/entry/613839 613839]. DHFRD is an inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency. Clinical features include variable neurologic symptoms, ranging from severe developmental delay and generalized seizures in infancy, to childhood absence epilepsy with learning difficulties, to lack of symptoms.<ref>PMID:21310276</ref> <ref>PMID:21310277</ref>  
== Function ==
== Function ==
[[http://www.uniprot.org/uniprot/DYR_HUMAN DYR_HUMAN]] Key enzyme in folate metabolism. Contributes to the de novo mitochondrial thymidylate biosynthesis pathway. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Binds its own mRNA and that of DHFRL1.<ref>PMID:21876188</ref> <ref>PMID:12096917</ref>
[https://www.uniprot.org/uniprot/DYR_HUMAN DYR_HUMAN] Key enzyme in folate metabolism. Contributes to the de novo mitochondrial thymidylate biosynthesis pathway. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Binds its own mRNA and that of DHFRL1.<ref>PMID:21876188</ref> <ref>PMID:12096917</ref>  
== Evolutionary Conservation ==
== Evolutionary Conservation ==
[[Image:Consurf_key_small.gif|200px|right]]
[[Image:Consurf_key_small.gif|200px|right]]
Check<jmol>
Check<jmol>
   <jmolCheckbox>
   <jmolCheckbox>
     <scriptWhenChecked>select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/dl/1dls_consurf.spt"</scriptWhenChecked>
     <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/dl/1dls_consurf.spt"</scriptWhenChecked>
     <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
     <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
     <text>to colour the structure by Evolutionary Conservation</text>
     <text>to colour the structure by Evolutionary Conservation</text>
   </jmolCheckbox>
   </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/chain_selection.php?pdb_ID=2ata ConSurf].
</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=1dls ConSurf].
<div style="clear:both"></div>
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.
Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.,Lewis WS, Cody V, Galitsky N, Luft JR, Pangborn W, Chunduru SK, Spencer HT, Appleman JR, Blakley RL J Biol Chem. 1995 Mar 10;270(10):5057-64. PMID:7890613<ref>PMID:7890613</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 1dls" style="background-color:#fffaf0;"></div>


==See Also==
==See Also==
*[[Dihydrofolate reductase|Dihydrofolate reductase]]
*[[Dihydrofolate reductase 3D structures|Dihydrofolate reductase 3D structures]]
== References ==
== References ==
<references/>
<references/>
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</StructureSection>
</StructureSection>
[[Category: Dihydrofolate Reductase]]
[[Category: Dihydrofolate Reductase]]
[[Category: Dihydrofolate reductase]]
[[Category: Homo sapiens]]
[[Category: Human]]
[[Category: Large Structures]]
[[Category: RCSB PDB Molecule of the Month]]
[[Category: RCSB PDB Molecule of the Month]]
[[Category: Cody, V]]
[[Category: Cody V]]
[[Category: Galitsky, N]]
[[Category: Galitsky N]]
[[Category: Luft, J R]]
[[Category: Luft JR]]
[[Category: Pangborn, W]]
[[Category: Pangborn W]]
[[Category: Oxido-reductase]]

Latest revision as of 09:55, 7 February 2024

METHOTREXATE-RESISTANT VARIANTS OF HUMAN DIHYDROFOLATE REDUCTASE WITH SUBSTITUTION OF LEUCINE 22: KINETICS, CRYSTALLOGRAPHY AND POTENTIAL AS SELECTABLE MARKERSMETHOTREXATE-RESISTANT VARIANTS OF HUMAN DIHYDROFOLATE REDUCTASE WITH SUBSTITUTION OF LEUCINE 22: KINETICS, CRYSTALLOGRAPHY AND POTENTIAL AS SELECTABLE MARKERS

Structural highlights

1dls is a 1 chain structure with sequence from Homo sapiens. The October 2002 RCSB PDB Molecule of the Month feature on Dihydrofolate Reductase by David S. Goodsell is 10.2210/rcsb_pdb/mom_2002_10. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 2.3Å
Ligands:,
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Disease

DYR_HUMAN Defects in DHFR are the cause of megaloblastic anemia due to dihydrofolate reductase deficiency (DHFRD) [MIM:613839. DHFRD is an inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency. Clinical features include variable neurologic symptoms, ranging from severe developmental delay and generalized seizures in infancy, to childhood absence epilepsy with learning difficulties, to lack of symptoms.[1] [2]

Function

DYR_HUMAN Key enzyme in folate metabolism. Contributes to the de novo mitochondrial thymidylate biosynthesis pathway. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Binds its own mRNA and that of DHFRL1.[3] [4]

Evolutionary Conservation

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

See Also

References

  1. ↑ Banka S, Blom HJ, Walter J, Aziz M, Urquhart J, Clouthier CM, Rice GI, de Brouwer AP, Hilton E, Vassallo G, Will A, Smith DE, Smulders YM, Wevers RA, Steinfeld R, Heales S, Crow YJ, Pelletier JN, Jones S, Newman WG. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency. Am J Hum Genet. 2011 Feb 11;88(2):216-25. doi: 10.1016/j.ajhg.2011.01.004. PMID:21310276 doi:10.1016/j.ajhg.2011.01.004
  2. ↑ Cario H, Smith DE, Blom H, Blau N, Bode H, Holzmann K, Pannicke U, Hopfner KP, Rump EM, Ayric Z, Kohne E, Debatin KM, Smulders Y, Schwarz K. Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease. Am J Hum Genet. 2011 Feb 11;88(2):226-31. doi: 10.1016/j.ajhg.2011.01.007. PMID:21310277 doi:10.1016/j.ajhg.2011.01.007
  3. ↑ Anderson DD, Quintero CM, Stover PJ. Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria. Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15163-8. doi:, 10.1073/pnas.1103623108. Epub 2011 Aug 26. PMID:21876188 doi:10.1073/pnas.1103623108
  4. ↑ Klon AE, Heroux A, Ross LJ, Pathak V, Johnson CA, Piper JR, Borhani DW. Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution. J Mol Biol. 2002 Jul 12;320(3):677-93. PMID:12096917

1dls, resolution 2.30Å

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