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New page: left|200px<br /><applet load="2gcb" size="450" color="white" frame="true" align="right" spinBox="true" caption="2gcb, resolution 2.3Å" /> '''G51S/S52T double muta...
 
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[[Image:2gcb.gif|left|200px]]<br /><applet load="2gcb" size="450" color="white" frame="true" align="right" spinBox="true"
caption="2gcb, resolution 2.3&Aring;" />
'''G51S/S52T double mutant of L. casei FPGS'''<br />


==Overview==
==G51S/S52T double mutant of L. casei FPGS==
Based upon the three-dimensional structure of Lactobacillus casei, folylpolyglutamate synthetase (FPGS), site-directed mutagenesis studies, were performed on three residues associated with the ATPase site: Gly51, Ser52 and Ser73. Gly51 and Ser52 are at the end of the P-loop, which is, involved in triphosphate binding. A G51S mutant enzyme and a G51S/S52T, double-mutant enzyme were made in order to alter the FPGS P-loop to more, closely resemble the sequences found in other ATPase and GTPase enzymes., Ser73 is on a neighboring loop (the Omega-loop) and precedes a proline, residue found to be in a cis conformation. The carbonyl O atom of Ser73 is, one of the protein ligands for the essential Mg(2+) ion involved in ATP, binding and hydrolysis and the Omega-loop is involved in binding the, folate substrate 5,10-methylenetetrahydrofolate. The serine residue was, mutated to alanine and this is the only one of the three mutants which, retains some FPGS activity. The structures of the G51S, G51S/S52T and S73A, mutant proteins have been solved to high resolution, along with the, structure of the apo wild-type FPGS. The P-loop in both the G51S and, G51S/S52T mutant proteins remains unaltered, yet both structures show a, large conformational rearrangement of the Omega-loop in which a cis-Pro, residue has switched conformation to a trans-peptide. The structure of the, Omega-loop is severely disrupted and as a consequence structural, rearrangements are observed in the peptide linker joining the two domains, of the enzyme. Magnesium binding in the active site is also disrupted by, the presence of the serine side chain at position 51 and by the, repositioning of the carbonyl O atom of Ser73 and a water molecule is, bound in place of the Mg(2+) ion. The S73A mutant protein retains the, cis-Pro configuration in the Omega-loop and the Mg(2+) site remains, intact. The cis-Pro is also observed in the structure of the, substrate-free form of FPGS (apoFPGS), maintained in the absence of Mg(2+), by a hydrogen-bonding network involving water molecules in the active, site. It is only in the complete absence of water or Mg(2+) in the binding, site that the cis-Pro switches to the trans conformation.
<StructureSection load='2gcb' size='340' side='right'caption='[[2gcb]], [[Resolution|resolution]] 2.30&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[2gcb]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Lacticaseibacillus_casei Lacticaseibacillus casei]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GCB OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2GCB 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]] 2.3&#8491;</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=2gcb FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2gcb OCA], [https://pdbe.org/2gcb PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2gcb RCSB], [https://www.ebi.ac.uk/pdbsum/2gcb PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2gcb ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/FPGS_LACCA FPGS_LACCA] Involved in the conversion of folates to polyglutamate derivatives, and likely functions in the retention of cellular folate pools. Catalyzes successive MgATP-dependent additions of glutamate to a pteroylmonoglutamate substrate, with a high preference for 5,10-methylenetetrahydrofolate (mTHF). Thus, metabolizes mTHF to the tetraglutamate derivative, but longer glutamate chain length products are not observed. Tetrahydrofolate (H4PteGlu) and 10-formyl-H4PteGlu are poorer folate substrates. In contrast to E.coli FolC, this enzyme does not display dihydrofolate synthase activity.<ref>PMID:18232714</ref> <ref>PMID:6138353</ref>
== 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/gc/2gcb_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=2gcb ConSurf].
<div style="clear:both"></div>


==About this Structure==
==See Also==
2GCB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Lactobacillus_casei Lactobacillus casei]. Active as [http://en.wikipedia.org/wiki/Tetrahydrofolate_synthase Tetrahydrofolate synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=6.3.2.17 6.3.2.17] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2GCB OCA].
*[[Folylpolyglutamate synthase|Folylpolyglutamate synthase]]
 
== References ==
==Reference==
<references/>
Mutation of Gly51 to serine in the P-loop of Lactobacillus casei folylpolyglutamate synthetase abolishes activity by altering the conformation of two adjacent loops., Smith CA, Cross JA, Bognar AL, Sun X, Acta Crystallogr D Biol Crystallogr. 2006 May;62(Pt 5):548-58. Epub 2006, Apr 19. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=16627949 16627949]
__TOC__
[[Category: Lactobacillus casei]]
</StructureSection>
[[Category: Single protein]]
[[Category: Lacticaseibacillus casei]]
[[Category: Tetrahydrofolate synthase]]
[[Category: Large Structures]]
[[Category: Bognar, A.L.]]
[[Category: Bognar AL]]
[[Category: Cross, J.A.]]
[[Category: Cross JA]]
[[Category: Smith, C.A.]]
[[Category: Smith CA]]
[[Category: Sun, X.]]
[[Category: Sun X]]
[[Category: atpase]]
[[Category: p-loop enzyme]]
[[Category: site-directed mutant]]
 
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 11:07:01 2007''

Latest revision as of 12:27, 14 February 2024

G51S/S52T double mutant of L. casei FPGSG51S/S52T double mutant of L. casei FPGS

Structural highlights

2gcb is a 1 chain structure with sequence from Lacticaseibacillus casei. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 2.3Å
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

FPGS_LACCA Involved in the conversion of folates to polyglutamate derivatives, and likely functions in the retention of cellular folate pools. Catalyzes successive MgATP-dependent additions of glutamate to a pteroylmonoglutamate substrate, with a high preference for 5,10-methylenetetrahydrofolate (mTHF). Thus, metabolizes mTHF to the tetraglutamate derivative, but longer glutamate chain length products are not observed. Tetrahydrofolate (H4PteGlu) and 10-formyl-H4PteGlu are poorer folate substrates. In contrast to E.coli FolC, this enzyme does not display dihydrofolate synthase activity.[1] [2]

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. ↑ Sheng Y, Khanam N, Tsaksis Y, Shi XM, Lu QS, Bognar AL. Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site. Biochemistry. 2008 Feb 26;47(8):2388-96. PMID:18232714 doi:10.1021/bi701670y
  2. ↑ Bognar AL, Shane B. Purification and properties of Lactobacillus casei folylpoly-gamma-glutamate synthetase. J Biol Chem. 1983 Oct 25;258(20):12574-81 PMID:6138353

2gcb, resolution 2.30Å

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