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| | ==X-ray Crystal Structure of a NADP(H)-bound Double Bond Reductase from Nicotiana tabacum== | | ==X-ray Crystal Structure of a NADP(H)-bound Double Bond Reductase from Nicotiana tabacum== |
| - | <StructureSection load='4hfm' size='340' side='right' caption='[[4hfm]], [[Resolution|resolution]] 1.90Å' scene=''> | + | <StructureSection load='4hfm' size='340' side='right'caption='[[4hfm]], [[Resolution|resolution]] 1.90Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[4hfm]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Nicotiana_tabacum Nicotiana tabacum]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4HFM OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4HFM FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[4hfm]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Nicotiana_tabacum Nicotiana tabacum]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4HFM OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=4HFM FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=NAP:NADP+NICOTINAMIDE-ADENINE-DINUCLEOTIDE+PHOSPHATE'>NAP</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]] 1.9Å</td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4hfj|4hfj]], [[4hfn|4hfn]]</td></tr>
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=NAP:NADP+NICOTINAMIDE-ADENINE-DINUCLEOTIDE+PHOSPHATE'>NAP</scene></td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">NtADH ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=4097 Nicotiana tabacum])</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=4hfm FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4hfm OCA], [https://pdbe.org/4hfm PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=4hfm RCSB], [https://www.ebi.ac.uk/pdbsum/4hfm PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=4hfm ProSAT]</span></td></tr> |
| - | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/2-alkenal_reductase 2-alkenal reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.1.74 1.3.1.74] </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=4hfm FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4hfm OCA], [http://www.rcsb.org/pdb/explore.do?structureId=4hfm RCSB], [http://www.ebi.ac.uk/pdbsum/4hfm PDBsum]</span></td></tr> | + | |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/Q9SLN8_TOBAC Q9SLN8_TOBAC]] Reduces the C=C double bonds of alpha, beta unsaturated enones, but has no activity on enones with an endocyclic C=C double-bond. Shows a high specificity for NADPH as the hybrid donor. Substrates are 1-nitrocyclohexene, 2-methylpentenal, trans-cinnamaldehyde, methyl-trans-2-methylcinnamaldehyde, trans-2-nonenal and 1-octen-3-one. Reduced activity with aplha-methyl transcinnamaldehyde, 1-cyclohexene-1-carboxaldehyde, methyl crotonate, (R)-pulegone, and dimethyl itaconate and no activity with maleimides, citral, (5R)- or (5S)-carvone, (S)-perillyl alcohol, and substituted cyclohexenones and cyclopentenones (PubMed:17945329, Ref.3). May also act as a allyl-alcohol dehydrogenase by catalyzing the dehydrogenation of secondary allylic alcohols rather than saturated secondary alcohols. Allyl-alcohol dehydrogenase is specific for the S-stereoisomer of the alcohols (PubMed:11117876).<ref>PMID:11117876</ref> <ref>PMID:17945329</ref> [REFERENCE:3] | + | [https://www.uniprot.org/uniprot/DBR_TOBAC DBR_TOBAC] Reduces the C=C double bonds of alpha, beta unsaturated enones, but has no activity on enones with an endocyclic C=C double-bond. Shows a high specificity for NADPH as the hybrid donor. Substrates are 1-nitrocyclohexene, 2-methylpentenal, trans-cinnamaldehyde, methyl-trans-2-methylcinnamaldehyde, trans-2-nonenal and 1-octen-3-one. Reduced activity with aplha-methyl transcinnamaldehyde, 1-cyclohexene-1-carboxaldehyde, methyl crotonate, (R)-pulegone, and dimethyl itaconate and no activity with maleimides, citral, (5R)- or (5S)-carvone, (S)-perillyl alcohol, and substituted cyclohexenones and cyclopentenones (PubMed:17945329, Ref.3). May also act as a allyl-alcohol dehydrogenase by catalyzing the dehydrogenation of secondary allylic alcohols rather than saturated secondary alcohols. Allyl-alcohol dehydrogenase is specific for the S-stereoisomer of the alcohols (PubMed:11117876).<ref>PMID:11117876</ref> <ref>PMID:17945329</ref> [REFERENCE:3] |
| | + | <div style="background-color:#fffaf0;"> |
| | + | == Publication Abstract from PubMed == |
| | + | The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of "-ene" reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover "-ene" reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of alpha,beta-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE "-ene" reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP(+) and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme. |
| | + | |
| | + | Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum.,Mansell DJ, Toogood HS, Waller J, Hughes JM, Levy CW, Gardiner JM, Scrutton NS ACS Catal. 2013 Mar 1;3(3):370-379. doi: 10.1021/cs300709m. Epub 2013 Jan 21. PMID:27547488<ref>PMID:27547488</ref> |
| | + | |
| | + | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
| | + | </div> |
| | + | <div class="pdbe-citations 4hfm" style="background-color:#fffaf0;"></div> |
| | | | |
| | ==See Also== | | ==See Also== |
| - | *[[Alcohol dehydrogenase|Alcohol dehydrogenase]] | + | *[[Alcohol dehydrogenase 3D structures|Alcohol dehydrogenase 3D structures]] |
| | == References == | | == References == |
| | <references/> | | <references/> |
| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: 2-alkenal reductase]] | + | [[Category: Large Structures]] |
| | [[Category: Nicotiana tabacum]] | | [[Category: Nicotiana tabacum]] |
| - | [[Category: Scrutton, N S]] | + | [[Category: Scrutton NS]] |
| - | [[Category: Toogood, H S]] | + | [[Category: Toogood HS]] |
| - | [[Category: Alkene reduction]]
| + | |
| - | [[Category: Oxidoreductase]]
| + | |
| - | [[Category: Rossmann fold]]
| + | |
| - | [[Category: Twisted b-barrel]]
| + | |
| Structural highlights
Function
DBR_TOBAC Reduces the C=C double bonds of alpha, beta unsaturated enones, but has no activity on enones with an endocyclic C=C double-bond. Shows a high specificity for NADPH as the hybrid donor. Substrates are 1-nitrocyclohexene, 2-methylpentenal, trans-cinnamaldehyde, methyl-trans-2-methylcinnamaldehyde, trans-2-nonenal and 1-octen-3-one. Reduced activity with aplha-methyl transcinnamaldehyde, 1-cyclohexene-1-carboxaldehyde, methyl crotonate, (R)-pulegone, and dimethyl itaconate and no activity with maleimides, citral, (5R)- or (5S)-carvone, (S)-perillyl alcohol, and substituted cyclohexenones and cyclopentenones (PubMed:17945329, Ref.3). May also act as a allyl-alcohol dehydrogenase by catalyzing the dehydrogenation of secondary allylic alcohols rather than saturated secondary alcohols. Allyl-alcohol dehydrogenase is specific for the S-stereoisomer of the alcohols (PubMed:11117876).[1] [2] [REFERENCE:3]
Publication Abstract from PubMed
The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of "-ene" reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover "-ene" reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of alpha,beta-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE "-ene" reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP(+) and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.
Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum.,Mansell DJ, Toogood HS, Waller J, Hughes JM, Levy CW, Gardiner JM, Scrutton NS ACS Catal. 2013 Mar 1;3(3):370-379. doi: 10.1021/cs300709m. Epub 2013 Jan 21. PMID:27547488[3]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Hirata T, Tamura Y, Yokobatake N, Shimoda K, Ashida Y. A 38 kDa allylic alcohol dehydrogenase from the cultured cells of Nicotiana tabacum. Phytochemistry. 2000 Oct;55(4):297-303. PMID:11117876
- ↑ Matsushima A, Sato Y, Otsuka M, Watanabe T, Yamamoto H, Hirata T. An enone reductase from Nicotiana tabacum: cDNA cloning, expression in Escherichia coli, and reduction of enones with the recombinant proteins. Bioorg Chem. 2008 Feb;36(1):23-8. Epub 2007 Oct 22. PMID:17945329 doi:http://dx.doi.org/10.1016/j.bioorg.2007.08.005
- ↑ Mansell DJ, Toogood HS, Waller J, Hughes JM, Levy CW, Gardiner JM, Scrutton NS. Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum. ACS Catal. 2013 Mar 1;3(3):370-379. doi: 10.1021/cs300709m. Epub 2013 Jan 21. PMID:27547488 doi:http://dx.doi.org/10.1021/cs300709m
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