1bwk
From Proteopedia
(New page: 200px<br /><applet load="1bwk" size="450" color="white" frame="true" align="right" spinBox="true" caption="1bwk, resolution 2.3Å" /> '''OLD YELLOW ENZYME (OY...) |
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| - | [[Image:1bwk.gif|left|200px]]<br /><applet load="1bwk" size=" | + | [[Image:1bwk.gif|left|200px]]<br /><applet load="1bwk" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1bwk, resolution 2.3Å" /> | caption="1bwk, resolution 2.3Å" /> | ||
'''OLD YELLOW ENZYME (OYE1) MUTANT H191N'''<br /> | '''OLD YELLOW ENZYME (OYE1) MUTANT H191N'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength | + | Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group. His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1. It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e. decreased binding affinity and decreased charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15. The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations. The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme. |
==About this Structure== | ==About this Structure== | ||
| - | 1BWK is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Candida_albicans Candida albicans] with FMN as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/NADPH_dehydrogenase NADPH dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.6.99.1 1.6.99.1] Full crystallographic information is available from [http:// | + | 1BWK is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Candida_albicans Candida albicans] with <scene name='pdbligand=FMN:'>FMN</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/NADPH_dehydrogenase NADPH dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.6.99.1 1.6.99.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1BWK OCA]. |
==Reference== | ==Reference== | ||
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[[Category: NADPH dehydrogenase]] | [[Category: NADPH dehydrogenase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Brown, B | + | [[Category: Brown, B J.]] |
[[Category: Deng, Z.]] | [[Category: Deng, Z.]] | ||
| - | [[Category: Karplus, P | + | [[Category: Karplus, P A.]] |
[[Category: Massey, V.]] | [[Category: Massey, V.]] | ||
[[Category: FMN]] | [[Category: FMN]] | ||
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[[Category: tim barrel]] | [[Category: tim barrel]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:59:53 2008'' |
Revision as of 09:59, 21 February 2008
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OLD YELLOW ENZYME (OYE1) MUTANT H191N
Overview
Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group. His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1. It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e. decreased binding affinity and decreased charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15. The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations. The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme.
About this Structure
1BWK is a Single protein structure of sequence from Candida albicans with as ligand. Active as NADPH dehydrogenase, with EC number 1.6.99.1 Full crystallographic information is available from OCA.
Reference
On the active site of Old Yellow Enzyme. Role of histidine 191 and asparagine 194., Brown BJ, Deng Z, Karplus PA, Massey V, J Biol Chem. 1998 Dec 4;273(49):32753-62. PMID:9830019
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