2dcy
From Proteopedia
(New page: 200px<br /><applet load="2dcy" size="450" color="white" frame="true" align="right" spinBox="true" caption="2dcy, resolution 1.40Å" /> '''Crystal structure of...) |
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| - | [[Image:2dcy.gif|left|200px]]<br /><applet load="2dcy" size=" | + | [[Image:2dcy.gif|left|200px]]<br /><applet load="2dcy" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2dcy, resolution 1.40Å" /> | caption="2dcy, resolution 1.40Å" /> | ||
'''Crystal structure of Bacillus subtilis family-11 xylanase'''<br /> | '''Crystal structure of Bacillus subtilis family-11 xylanase'''<br /> | ||
==Overview== | ==Overview== | ||
| - | We used directed evolution to enhance the thermostability of glycosyl | + | We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xyl(st), was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xyl(st) variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 degrees C. At 60 degrees C the wild-type enzyme was inactivated within 5 min, but Xyl(st) retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 degrees C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xyl(st) enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xyl(st). This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xyl(st). However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability. |
==About this Structure== | ==About this Structure== | ||
| - | 2DCY is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis] with TAR and DIO as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Endo-1,4-beta-xylanase Endo-1,4-beta-xylanase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.8 3.2.1.8] Full crystallographic information is available from [http:// | + | 2DCY is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis] with <scene name='pdbligand=TAR:'>TAR</scene> and <scene name='pdbligand=DIO:'>DIO</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Endo-1,4-beta-xylanase Endo-1,4-beta-xylanase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.8 3.2.1.8] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2DCY OCA]. |
==Reference== | ==Reference== | ||
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[[Category: all beta]] | [[Category: all beta]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:57:34 2008'' |
Revision as of 14:57, 21 February 2008
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Crystal structure of Bacillus subtilis family-11 xylanase
Overview
We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xyl(st), was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xyl(st) variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 degrees C. At 60 degrees C the wild-type enzyme was inactivated within 5 min, but Xyl(st) retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 degrees C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xyl(st) enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xyl(st). This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xyl(st). However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability.
About this Structure
2DCY is a Single protein structure of sequence from Bacillus subtilis with and as ligands. Active as Endo-1,4-beta-xylanase, with EC number 3.2.1.8 Full crystallographic information is available from OCA.
Reference
Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution., Miyazaki K, Takenouchi M, Kondo H, Noro N, Suzuki M, Tsuda S, J Biol Chem. 2006 Apr 14;281(15):10236-42. Epub 2006 Feb 8. PMID:16467302
Page seeded by OCA on Thu Feb 21 16:57:34 2008
Categories: Bacillus subtilis | Endo-1,4-beta-xylanase | Single protein | Kondo, H. | Miyazaki, K. | Noro, N. | Suzuki, M. | Takenouchi, M. | Tsuda, S. | DIO | TAR | All beta
