2dcy
From Proteopedia
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| - | + | {{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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[[Category: Takenouchi, M.]] | [[Category: Takenouchi, M.]] | ||
[[Category: Tsuda, S.]] | [[Category: Tsuda, S.]] | ||
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Revision as of 21:11, 3 May 2008
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. 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 Sun May 4 00:11:36 2008
