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1jkx

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[[Image:1jkx.gif|left|200px]]<br /><applet load="1jkx" size="350" color="white" frame="true" align="right" spinBox="true"
 
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caption="1jkx, resolution 1.60&Aring;" />
 
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'''Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase'''<br />
 
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==Overview==
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==Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase==
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Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transformylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range. However, these compounds have reduced bioavailability due to the incorporation of a negatively charged phosphate moiety that prevents effective cellular uptake. Thus, a folate analogue that is capable of adduct formation with the substrate on the enzyme active site could lead to a potent GAR Tfase inhibitor that takes advantage of the cellular folate transport systems. We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an intermediate designed to assemble with the substrate beta-GAR on the enzyme active site. We have now determined the crystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism of its time-dependent inhibition. The high-resolution crystal structure clearly revealed the existence of a covalent adduct between the substrate beta-GAR and the folate analogue (K(i) = 20 microM). However, the electron density map surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but proceeds via an epoxide. Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide. Further, inhibition was strongest when the dibromide or epoxide is preincubated with both enzyme and substrate, indicating that inhibition occurs via the enzyme-dependent formation of the multisubstrate adduct. Thus, the crystal structure revealed the successful formation of an enzyme-assembled multisubstrate adduct and highlighted a potential application for epoxides, and perhaps aziridines, in the design of efficacious GAR Tfase inhibitors.
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<StructureSection load='1jkx' size='340' side='right'caption='[[1jkx]], [[Resolution|resolution]] 1.60&Aring;' scene=''>
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== Structural highlights ==
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==About this Structure==
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<table><tr><td colspan='2'>[[1jkx]] is a 4 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=1JKX OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1JKX FirstGlance]. <br>
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1JKX 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=138:'>138</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Phosphoribosylglycinamide_formyltransferase Phosphoribosylglycinamide formyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.2.2 2.1.2.2] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1JKX OCA].
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</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.6&#8491;</td></tr>
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<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=138:N-[5-O-PHOSPHONO-RIBOFURANOSYL]-2-[2-HYDROXY-2-[4-[GLUTAMIC+ACID]-N-CARBONYLPHENYL]-3-[2-AMINO-4-HYDROXY-QUINAZOLIN-6-YL]-PROPANYLAMINO]-ACETAMIDE'>138</scene></td></tr>
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==Reference==
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<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1jkx FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1jkx OCA], [https://pdbe.org/1jkx PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1jkx RCSB], [https://www.ebi.ac.uk/pdbsum/1jkx PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1jkx ProSAT]</span></td></tr>
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Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase., Greasley SE, Marsilje TH, Cai H, Baker S, Benkovic SJ, Boger DL, Wilson IA, Biochemistry. 2001 Nov 13;40(45):13538-47. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=11695901 11695901]
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</table>
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== Function ==
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[https://www.uniprot.org/uniprot/PUR3_ECOLI PUR3_ECOLI]
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== Evolutionary Conservation ==
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[[Image:Consurf_key_small.gif|200px|right]]
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Check<jmol>
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<jmolCheckbox>
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<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/jk/1jkx_consurf.spt"</scriptWhenChecked>
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<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
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<text>to colour the structure by Evolutionary Conservation</text>
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</jmolCheckbox>
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</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=1jkx ConSurf].
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<div style="clear:both"></div>
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__TOC__
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</StructureSection>
[[Category: Escherichia coli]]
[[Category: Escherichia coli]]
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[[Category: Phosphoribosylglycinamide formyltransferase]]
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[[Category: Large Structures]]
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[[Category: Single protein]]
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[[Category: Baker S]]
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[[Category: Baker, S.]]
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[[Category: Benkovic SJ]]
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[[Category: Benkovic, S J.]]
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[[Category: Boger DL]]
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[[Category: Boger, D L.]]
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[[Category: Cai H]]
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[[Category: Cai, H.]]
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[[Category: Greasley SE]]
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[[Category: Greasley, S E.]]
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[[Category: Marsilje TH]]
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[[Category: Marsilje, T H.]]
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[[Category: Wilson IA]]
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[[Category: Wilson, I A.]]
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[[Category: 138]]
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[[Category: anti-cancer agent]]
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[[Category: enzyme-assembled multisubstrate adduct inhibitor complex]]
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[[Category: purine biosynthesis]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:23:48 2008''
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Current revision

Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase

PDB ID 1jkx

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