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| | <StructureSection load='3ah3' size='340' side='right'caption='[[3ah3]], [[Resolution|resolution]] 2.40Å' scene=''> | | <StructureSection load='3ah3' size='340' side='right'caption='[[3ah3]], [[Resolution|resolution]] 2.40Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[3ah3]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Thet2 Thet2]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3AH3 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3AH3 FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[3ah3]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Thermus_thermophilus_HB27 Thermus thermophilus HB27]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3AH3 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3AH3 FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</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]] 2.4Å</td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat"><div style='overflow: auto; max-height: 3em;'>[[1x0l|1x0l]]</div></td></tr>
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">LR5-1 ([https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=262724 THET2])</td></tr> | + | |
| - | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[https://en.wikipedia.org/wiki/Homoisocitrate_dehydrogenase Homoisocitrate dehydrogenase], with EC number [https://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.87 1.1.1.87] </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=3ah3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3ah3 OCA], [https://pdbe.org/3ah3 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3ah3 RCSB], [https://www.ebi.ac.uk/pdbsum/3ah3 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3ah3 ProSAT]</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=3ah3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3ah3 OCA], [https://pdbe.org/3ah3 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3ah3 RCSB], [https://www.ebi.ac.uk/pdbsum/3ah3 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3ah3 ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[https://www.uniprot.org/uniprot/HICDH_THET2 HICDH_THET2]] Catalyzes the NAD(+)-dependent conversion of homoisocitrate to alpha-ketoadipate. In addition, has high activity with citrate, but is inactive with 3-isopropylmalate.<ref>PMID:12427751</ref> <ref>PMID:20735360</ref>
| + | [https://www.uniprot.org/uniprot/HICDH_THET2 HICDH_THET2] Catalyzes the NAD(+)-dependent conversion of homoisocitrate to alpha-ketoadipate. In addition, has high activity with citrate, but is inactive with 3-isopropylmalate.<ref>PMID:12427751</ref> <ref>PMID:20735360</ref> |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
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| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Homoisocitrate dehydrogenase]] | |
| | [[Category: Large Structures]] | | [[Category: Large Structures]] |
| - | [[Category: Thet2]] | + | [[Category: Thermus thermophilus HB27]] |
| - | [[Category: Kuzuyama, T]] | + | [[Category: Kuzuyama T]] |
| - | [[Category: Nishiyama, M]] | + | [[Category: Nishiyama M]] |
| - | [[Category: Suzuki, Y]] | + | [[Category: Suzuki Y]] |
| - | [[Category: Tomita, T]] | + | [[Category: Tomita T]] |
| - | [[Category: 3-isopropylmalate dehydrogenase]]
| + | |
| - | [[Category: Directed evolution]]
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| - | [[Category: Oxidoreductase]]
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| Structural highlights
Function
HICDH_THET2 Catalyzes the NAD(+)-dependent conversion of homoisocitrate to alpha-ketoadipate. In addition, has high activity with citrate, but is inactive with 3-isopropylmalate.[1] [2]
Publication Abstract from PubMed
HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through alpha-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB) to grow on a minimal medium in 2 days were obtained. One of the variants LR5-1, with eight amino acid replacements, was found to possess a 65-fold increased k(cat)/K(m) value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the k(cat)/K(m) value and a partial recovery of the decreased thermotolerance of LR5-1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5-1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5-1 was determined at 2.4 A resolution and revealed that helix alpha4 was displaced in a manner suitable for recognition of the hydrophobic gamma-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.
Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution.,Suzuki Y, Asada K, Miyazaki J, Tomita T, Kuzuyama T, Nishiyama M Biochem J. 2010 Oct 11;431(3):401-10. PMID:20735360[3]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Miyazaki J, Kobashi N, Nishiyama M, Yamane H. Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium, Thermus thermophilus HB27, and evolutionary implication of beta-decarboxylating dehydrogenase. J Biol Chem. 2003 Jan 17;278(3):1864-71. Epub 2002 Nov 8. PMID:12427751 doi:http://dx.doi.org/10.1074/jbc.M205133200
- ↑ Suzuki Y, Asada K, Miyazaki J, Tomita T, Kuzuyama T, Nishiyama M. Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution. Biochem J. 2010 Oct 11;431(3):401-10. PMID:20735360 doi:10.1042/BJ20101246
- ↑ Suzuki Y, Asada K, Miyazaki J, Tomita T, Kuzuyama T, Nishiyama M. Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution. Biochem J. 2010 Oct 11;431(3):401-10. PMID:20735360 doi:10.1042/BJ20101246
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