4K6G

Crystal structure of CALB from Candida antarctica

4K6G の概要

• エントリーDOI: 10.2210/pdb4k6g/pdb
• 関連するPDBエントリー: 4K5Q 4K6H 4K6K
• 分子名称: Lipase B, 1,2-ETHANEDIOL (3 entities in total)
• 機能のキーワード: lipase, hydrolase
• 由来する生物種: Candida antarctica (Yeast)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 68751.43

構造登録者

An, J.,Xie, Y.,Feng, Y.,Wu, G. (登録日: 2013-04-15, 公開日: 2014-01-29, 最終更新日: 2024-10-16)

主引用文献

Xie, Y.,An, J.,Yang, G.,Wu, G.,Zhang, Y.,Cui, L.,Feng, Y.
Enhanced enzyme kinetic stability by increasing rigidity within the active site.
J.Biol.Chem., 289:7994-8006, 2014

PubMed Abstract: Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

PubMed: 24448805
DOI: 10.1074/jbc.M113.536045

実験手法

X-RAY DIFFRACTION (1.5 Å)