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| | <StructureSection load='3vzs' size='340' side='right'caption='[[3vzs]], [[Resolution|resolution]] 2.14Å' scene=''> | | <StructureSection load='3vzs' size='340' side='right'caption='[[3vzs]], [[Resolution|resolution]] 2.14Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[3vzs]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Alcaligenes_eutropha_h16 Alcaligenes eutropha h16]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3VZS OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3VZS FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[3vzs]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Cupriavidus_necator_H16 Cupriavidus necator H16]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3VZS OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3VZS FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CAA:ACETOACETYL-COENZYME+A'>CAA</scene>, <scene name='pdbligand=NAP:NADP+NICOTINAMIDE-ADENINE-DINUCLEOTIDE+PHOSPHATE'>NAP</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]] 2.14Å</td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat"><div style='overflow: auto; max-height: 3em;'>[[3vzp|3vzp]], [[3vzq|3vzq]], [[3vzr|3vzr]]</div></td></tr> | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CAA:ACETOACETYL-COENZYME+A'>CAA</scene>, <scene name='pdbligand=NAP:NADP+NICOTINAMIDE-ADENINE-DINUCLEOTIDE+PHOSPHATE'>NAP</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">phbB, H16_A1439 ([https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=381666 Alcaligenes eutropha H16])</td></tr>
| + | |
| - | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[https://en.wikipedia.org/wiki/Acetoacetyl-CoA_reductase Acetoacetyl-CoA reductase], with EC number [https://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.36 1.1.1.36] </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=3vzs FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3vzs OCA], [https://pdbe.org/3vzs PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3vzs RCSB], [https://www.ebi.ac.uk/pdbsum/3vzs PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3vzs 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=3vzs FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3vzs OCA], [https://pdbe.org/3vzs PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3vzs RCSB], [https://www.ebi.ac.uk/pdbsum/3vzs PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3vzs ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | + | == Function == |
| | + | [https://www.uniprot.org/uniprot/PHAB_CUPNH PHAB_CUPNH] Catalyzes the chiral reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Is involved in the biosynthesis of polyhydroxybutyrate (PHB), which is accumulated as an intracellular energy reserve material when cells grow under conditions of nutrient limitation.<ref>PMID:23913421</ref> <ref>PMID:24211201</ref> <ref>PMID:2670935</ref> <ref>PMID:2670936</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: Acetoacetyl-CoA reductase]] | + | [[Category: Cupriavidus necator H16]] |
| - | [[Category: Alcaligenes eutropha h16]]
| + | |
| | [[Category: Large Structures]] | | [[Category: Large Structures]] |
| - | [[Category: Ikeda, K]] | + | [[Category: Ikeda K]] |
| - | [[Category: Tanaka, I]] | + | [[Category: Tanaka I]] |
| - | [[Category: Tanaka, Y]] | + | [[Category: Tanaka Y]] |
| - | [[Category: Yao, M]] | + | [[Category: Yao M]] |
| - | [[Category: Alpha/beta fold]]
| + | |
| - | [[Category: Oxidoreductase]]
| + | |
| Structural highlights
Function
PHAB_CUPNH Catalyzes the chiral reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Is involved in the biosynthesis of polyhydroxybutyrate (PHB), which is accumulated as an intracellular energy reserve material when cells grow under conditions of nutrient limitation.[1] [2] [3] [4]
Publication Abstract from PubMed
Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent acetoacetyl-CoA reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with beta-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. Out of approximately twenty thousand mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants respectively exhibited a 2.4 and 3.5-fold higher kcat value compared to that of wild-type enzyme. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis between the wild-type and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn play an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
Directed evolution and structural analysis of NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.,Matsumoto K, Tanaka Y, Watanabe T, Motohashi R, Ikeda K, Tobitani K, Yao M, Tanaka I, Taguchi S Appl Environ Microbiol. 2013 Aug 2. PMID:23913421[5]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Matsumoto K, Tanaka Y, Watanabe T, Motohashi R, Ikeda K, Tobitani K, Yao M, Tanaka I, Taguchi S. Directed evolution and structural analysis of NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics. Appl Environ Microbiol. 2013 Aug 2. PMID:23913421 doi:10.1128/AEM.01768-13
- ↑ Kim J, Chang JH, Kim EJ, Kim KJ. Crystal structure of (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha. Biochem Biophys Res Commun. 2013 Nov 6. pii: S0006-291X(13)01843-3. doi:, 10.1016/j.bbrc.2013.10.150. PMID:24211201 doi:http://dx.doi.org/10.1016/j.bbrc.2013.10.150
- ↑ Peoples OP, Sinskey AJ. Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase. J Biol Chem. 1989 Sep 15;264(26):15293-7. PMID:2670935
- ↑ Peoples OP, Sinskey AJ. Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). J Biol Chem. 1989 Sep 15;264(26):15298-303. PMID:2670936
- ↑ Matsumoto K, Tanaka Y, Watanabe T, Motohashi R, Ikeda K, Tobitani K, Yao M, Tanaka I, Taguchi S. Directed evolution and structural analysis of NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics. Appl Environ Microbiol. 2013 Aug 2. PMID:23913421 doi:10.1128/AEM.01768-13
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