6ISP

structure of Candida antarctica Lipase B mutant

6ISP の概要

• エントリーDOI: 10.2210/pdb6isp/pdb
• 分子名称: Lipase B, N,N-BIS(3-D-GLUCONAMIDOPROPYL)DEOXYCHOLAMIDE, CALCIUM ION, ... (4 entities in total)
• 機能のキーワード: calb, hydrolase
• 由来する生物種: Pseudozyma antarctica (Yeast)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 143265.46

構造登録者

Cen, Y.X.,Zhou, J.H.,Wu, Q. (登録日: 2018-11-18, 公開日: 2019-07-24, 最終更新日: 2024-10-16)

主引用文献

Cen, Y.,Singh, W.,Arkin, M.,Moody, T.S.,Huang, M.,Zhou, J.,Wu, Q.,Reetz, M.T.
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.
Nat Commun, 10:3198-3198, 2019

PubMed Abstract: Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105/His224 rather than a concerted process.

PubMed: 31324776
DOI: 10.1038/s41467-019-11155-3

実験手法

X-RAY DIFFRACTION (1.88 Å)