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| - | [[Image:2dcy.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_2dcy| PDB=2dcy | SCENE= }} | | {{STRUCTURE_2dcy| PDB=2dcy | SCENE= }} |
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| - | '''Crystal structure of Bacillus subtilis family-11 xylanase'''
| + | ===Crystal structure of Bacillus subtilis family-11 xylanase=== |
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| - | ==Overview==
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| - | 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_16467302}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 16467302 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_16467302}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Tsuda, S.]] | | [[Category: Tsuda, S.]] |
| | [[Category: All beta]] | | [[Category: All beta]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 00:11:36 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Jul 27 19:40:54 2008'' |
Revision as of 16:40, 27 July 2008
Template:STRUCTURE 2dcy
Crystal structure of Bacillus subtilis family-11 xylanase
Template:ABSTRACT PUBMED 16467302
About this Structure
2DCY is a Single protein structure of sequence from Bacillus subtilis. 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
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