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| - | [[Image:2glx.gif|left|200px]] | |
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| - | {{Structure
| + | ==Crystal Structure Analysis of bacterial 1,5-AF Reductase== |
| - | |PDB= 2glx |SIZE=350|CAPTION= <scene name='initialview01'>2glx</scene>, resolution 2.200Å
| + | <StructureSection load='2glx' size='340' side='right'caption='[[2glx]], [[Resolution|resolution]] 2.20Å' scene=''> |
| - | |SITE=
| + | == Structural highlights == |
| - | |LIGAND= <scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene> and <scene name='pdbligand=NDP:NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE'>NDP</scene> | + | <table><tr><td colspan='2'>[[2glx]] is a 6 chain structure with sequence from [https://en.wikipedia.org/wiki/Ensifer_adhaerens Ensifer adhaerens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GLX OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2GLX FirstGlance]. <br> |
| - | |ACTIVITY= [http://en.wikipedia.org/wiki/1,5-anhydro-D-fructose_reductase 1,5-anhydro-D-fructose reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.263 1.1.1.263]
| + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.2Å</td></tr> |
| - | |GENE= afr ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=106592 Ensifer adhaerens])
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=NDP:NADPH+DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE+PHOSPHATE'>NDP</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=2glx FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2glx OCA], [https://pdbe.org/2glx PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2glx RCSB], [https://www.ebi.ac.uk/pdbsum/2glx PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2glx ProSAT]</span></td></tr> |
| - | | + | </table> |
| - | '''Crystal Structure Analysis of bacterial 1,5-AF Reductase'''
| + | == Function == |
| - | | + | [https://www.uniprot.org/uniprot/AFR_ENSAD AFR_ENSAD] Catalyzes the NADPH-specific reduction of 1,5-anhydro-D-fructose to 1,5-anhydro-D-mannitol. Also shows some activity against structurally related compounds such as 3-keto-1,5-anhydro-D-fructose, D-glucosone and D-xylosone. The enzyme cannot use NADH as cosubstrate. |
| - | | + | == Evolutionary Conservation == |
| - | ==Overview== | + | [[Image:Consurf_key_small.gif|200px|right]] |
| - | Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium morelense S-30.7.5 was crystallized in complex with the cofactor NADP(H) and its structure determined to 2.2 A resolution using selenomethionine SAD (refined R(work) and R(free) factors of 18.9 and 25.0%, respectively). As predicted from the sequence and shown by the structure, AFR can be assigned to the GFO/IDH/MocA protein family. AFR consists of two domains. The N-terminal domain displays a Rossmann fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP(+), whose attachment to the cofactor binding site is similar to that of NADP(+) in glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. Due to variations in length and sequence within loop regions L3 and L5, respectively, the adenine moiety of NADP(+) adopts a different orientation in AFR caused by residue Arg38 forming hydrogen bonds with the 2'-phosphate moiety of NADP(+) and cation-pi stacking interactions with the adenine ring. Amino acid replacements in AFR (S10G, A13G, and S33D) showed that Ala13 is crucial for the discrimination between NADPH and NADH and yielded the A13G variant with dual cosubstrate specificity. The C-terminal domain contains the putative substrate binding site that was occupied by an acetate ion. As determined by analogy to GFOR and by site-directed mutagenesis of K94G, D176A, and H180A, residues Lys94, Asp176, and His180 are most likely involved in substrate binding and catalysis, as substitution of any of these residues resulted in a significant decrease in k(cat) for 1,5-AF. In this context, His180 might serve as a general acid-base catalyst by polarizing the carbonyl function of 1,5-AF to enable the transfer of the hydride from NADPH to the substrate. Here we present the first structure of an AFR enzyme catalyzing the stereoselective reduction of 1,5-AF to 1,5-anhydro-d-mannitol, the final step of a modified anhydrofructose pathway in S. morelense S-30.7.5. We also emphasize the importance of the A13G variant in biocatalysis for the synthesis of 1,5-AM and related derivatives.
| + | Check<jmol> |
| - | | + | <jmolCheckbox> |
| - | ==About this Structure== | + | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/gl/2glx_consurf.spt"</scriptWhenChecked> |
| - | 2GLX is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Ensifer_adhaerens Ensifer adhaerens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GLX OCA].
| + | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> |
| - | | + | <text>to colour the structure by Evolutionary Conservation</text> |
| - | ==Reference==
| + | </jmolCheckbox> |
| - | Crystal structure of NADP(H)-dependent 1,5-anhydro-D-fructose reductase from Sinorhizobium morelense at 2.2 A resolution: construction of a NADH-accepting mutant and its application in rare sugar synthesis., Dambe TR, Kuhn AM, Brossette T, Giffhorn F, Scheidig AJ, Biochemistry. 2006 Aug 22;45(33):10030-42. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16906761 16906761]
| + | </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=2glx ConSurf]. |
| - | [[Category: 1,5-anhydro-D-fructose reductase]]
| + | <div style="clear:both"></div> |
| | + | __TOC__ |
| | + | </StructureSection> |
| | [[Category: Ensifer adhaerens]] | | [[Category: Ensifer adhaerens]] |
| - | [[Category: Single protein]] | + | [[Category: Large Structures]] |
| - | [[Category: Dambe, T R.]] | + | [[Category: Dambe TR]] |
| - | [[Category: Scheidig, A J.]] | + | [[Category: Scheidig AJ]] |
| - | [[Category: ACT]]
| + | |
| - | [[Category: NDP]]
| + | |
| - | [[Category: 1]]
| + | |
| - | [[Category: 5-anhydro-d-fructose]]
| + | |
| - | [[Category: 5-anhydro-d-fructose reductase]]
| + | |
| - | [[Category: 5-anhydro-d-mannitol]]
| + | |
| - | [[Category: bacterial 1]]
| + | |
| - | [[Category: nadp(h) dependent reductase]]
| + | |
| - | [[Category: rossmann-fold]]
| + | |
| - | [[Category: sugar metabolism]]
| + | |
| - | | + | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 17:06:15 2008''
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