2DCY

Crystal structure of Bacillus subtilis family-11 xylanase

Summary for 2DCY

• Entry DOI: 10.2210/pdb2dcy/pdb
• Related: 2DCZ
• Descriptor: Endo-1,4-beta-xylanase A, 1,4-DIETHYLENE DIOXIDE, L(+)-TARTARIC ACID, ... (5 entities in total)
• Functional Keywords: all beta, hydrolase
• Biological source: Bacillus subtilis
• Total number of polymer chains: 5
• Total formula weight: 103289.82

Authors

Kondo, H.,Miyazaki, K.,Takenouchi, M.,Noro, N.,Suzuki, M.,Tsuda, S. (deposition date: 2006-01-18, release date: 2006-02-07, Last modification date: 2023-10-25)

Primary citation

Miyazaki, K.,Takenouchi, M.,Kondo, H.,Noro, N.,Suzuki, M.,Tsuda, S.
Thermal Stabilization of Bacillus subtilis Family-11 Xylanase by Directed Evolution
J.Biol.Chem., 281:10236-10242, 2006

PubMed Abstract: 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.

PubMed: 16467302
DOI: 10.1074/jbc.M511948200

Experimental method

X-RAY DIFFRACTION (1.4 Å)