1t2x
From Proteopedia
(New page: 200px<br /><applet load="1t2x" size="450" color="white" frame="true" align="right" spinBox="true" caption="1t2x, resolution 2.3Å" /> '''Glactose oxidase C383...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:1t2x.gif|left|200px]]<br /><applet load="1t2x" size=" | + | [[Image:1t2x.gif|left|200px]]<br /><applet load="1t2x" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1t2x, resolution 2.3Å" /> | caption="1t2x, resolution 2.3Å" /> | ||
'''Glactose oxidase C383S mutant identified by directed evolution'''<br /> | '''Glactose oxidase C383S mutant identified by directed evolution'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that | + | Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that oxidizes a range of primary alcohols to aldehydes. This broad substrate specificity is reflected in a high K(M) for substrates. Directed evolution has previously been used to select variants of GO that exhibit enhanced expression and kinetic properties. In assays using unpurified enzyme samples, the variant C383S displayed a 5-fold lower K(M) than wild-type GO. In the present study, we have constructed, expressed, purified and characterized a number of single, double and triple mutants at residues Cys383, Tyr436 and Val494, identified in one of the directed evolution studies, to examine their relative contributions to improved catalytic activity of GO. We report kinetic studies on the various mutant enzymes. In addition, we have determined the three-dimensional structure of the C383S variant. As with many mutations identified in directed evolution experiments, the availability of structural information does not provide a definitive answer to the reason for the improved K(M) in the C383S variant protein. |
==About this Structure== | ==About this Structure== | ||
| - | 1T2X is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Fusarium_sp. Fusarium sp.] with NA, CU and ACT as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Galactose_oxidase Galactose oxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.9 1.1.3.9] Full crystallographic information is available from [http:// | + | 1T2X is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Fusarium_sp. Fusarium sp.] with <scene name='pdbligand=NA:'>NA</scene>, <scene name='pdbligand=CU:'>CU</scene> and <scene name='pdbligand=ACT:'>ACT</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Galactose_oxidase Galactose oxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.9 1.1.3.9] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1T2X OCA]. |
==Reference== | ==Reference== | ||
| Line 17: | Line 17: | ||
[[Category: Dawkes, H.]] | [[Category: Dawkes, H.]] | ||
[[Category: Hurtado-Guerrero, R.]] | [[Category: Hurtado-Guerrero, R.]] | ||
| - | [[Category: Knowles, P | + | [[Category: Knowles, P F.]] |
| - | [[Category: McPherson, M | + | [[Category: McPherson, M J.]] |
| - | [[Category: Phillips, S | + | [[Category: Phillips, S E.V.]] |
[[Category: Wilkinson, D.]] | [[Category: Wilkinson, D.]] | ||
[[Category: ACT]] | [[Category: ACT]] | ||
| Line 28: | Line 28: | ||
[[Category: mutant form of copper containing enzyme]] | [[Category: mutant form of copper containing enzyme]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:09:17 2008'' |
Revision as of 13:09, 21 February 2008
|
Glactose oxidase C383S mutant identified by directed evolution
Overview
Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that oxidizes a range of primary alcohols to aldehydes. This broad substrate specificity is reflected in a high K(M) for substrates. Directed evolution has previously been used to select variants of GO that exhibit enhanced expression and kinetic properties. In assays using unpurified enzyme samples, the variant C383S displayed a 5-fold lower K(M) than wild-type GO. In the present study, we have constructed, expressed, purified and characterized a number of single, double and triple mutants at residues Cys383, Tyr436 and Val494, identified in one of the directed evolution studies, to examine their relative contributions to improved catalytic activity of GO. We report kinetic studies on the various mutant enzymes. In addition, we have determined the three-dimensional structure of the C383S variant. As with many mutations identified in directed evolution experiments, the availability of structural information does not provide a definitive answer to the reason for the improved K(M) in the C383S variant protein.
About this Structure
1T2X is a Protein complex structure of sequences from Fusarium sp. with , and as ligands. Active as Galactose oxidase, with EC number 1.1.3.9 Full crystallographic information is available from OCA.
Reference
Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution., Wilkinson D, Akumanyi N, Hurtado-Guerrero R, Dawkes H, Knowles PF, Phillips SE, McPherson MJ, Protein Eng Des Sel. 2004 Feb;17(2):141-8. Epub 2004 Jan 12. PMID:15047910
Page seeded by OCA on Thu Feb 21 15:09:17 2008
