4zf6: Difference between revisions
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The | ==Cytochrome P450 pentamutant from BM3 with bound PEG== | ||
<StructureSection load='4zf6' size='340' side='right' caption='[[4zf6]], [[Resolution|resolution]] 2.77Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[4zf6]] is a 1 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4ZF6 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4ZF6 FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=1PE:PENTAETHYLENE+GLYCOL'>1PE</scene>, <scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene>, <scene name='pdbligand=NI:NICKEL+(II)+ION'>NI</scene></td></tr> | |||
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4zf8|4zf8]], [[4zf9|4zf9]], [[4zfa|4zfa]], [[4zfb|4zfb]], [[4zfd|4zfd]], [[4zfe|4zfe]]</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=4zf6 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4zf6 OCA], [http://pdbe.org/4zf6 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=4zf6 RCSB], [http://www.ebi.ac.uk/pdbsum/4zf6 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=4zf6 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[[http://www.uniprot.org/uniprot/CPXB_BACME CPXB_BACME]] Functions as a fatty acid monooxygenase. Catalyzes hydroxylation of medium and long-chain fatty acids at omega-1, omega-2 and omega-3 positions, with optimum chain lengths of 12-16 carbons (lauric, myristic, and palmitic acids). The reductase domain is required for electron transfer from NADP to cytochrome P450. | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Cytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, beta-sheet 1, and Cys ligand loop (a beta-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into beta-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability. | |||
Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.,Geronimo I, Denning CA, Rogers WE, Othman T, Huxford T, Heidary DK, Glazer EC, Payne CM Biochemistry. 2016 Jun 28;55(25):3594-606. doi: 10.1021/acs.biochem.6b00183. Epub, 2016 Jun 16. PMID:27267136<ref>PMID:27267136</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
<div class="pdbe-citations 4zf6" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Heidary, D K]] | |||
[[Category: Huxford, T]] | [[Category: Huxford, T]] | ||
[[Category: Othman, T]] | [[Category: Othman, T]] | ||
[[Category: Rogers, W | [[Category: Rogers, W E]] | ||
[[Category: Bacillus megaterium]] | |||
[[Category: Cytochrome p450]] | |||
[[Category: Heme oxidase domain]] | |||
[[Category: Oxidoreductase]] | |||
Revision as of 14:40, 14 July 2016
Cytochrome P450 pentamutant from BM3 with bound PEGCytochrome P450 pentamutant from BM3 with bound PEG
Structural highlights
Function[CPXB_BACME] Functions as a fatty acid monooxygenase. Catalyzes hydroxylation of medium and long-chain fatty acids at omega-1, omega-2 and omega-3 positions, with optimum chain lengths of 12-16 carbons (lauric, myristic, and palmitic acids). The reductase domain is required for electron transfer from NADP to cytochrome P450. Publication Abstract from PubMedCytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, beta-sheet 1, and Cys ligand loop (a beta-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into beta-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability. Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.,Geronimo I, Denning CA, Rogers WE, Othman T, Huxford T, Heidary DK, Glazer EC, Payne CM Biochemistry. 2016 Jun 28;55(25):3594-606. doi: 10.1021/acs.biochem.6b00183. Epub, 2016 Jun 16. PMID:27267136[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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