4P08

Engineered thermostable dimeric cocaine esterase

4P08 の概要

• エントリーDOI: 10.2210/pdb4p08/pdb
• 分子名称: Cocaine esterase (2 entities in total)
• 機能のキーワード: esterase, disulfide-linked dimer, cocaine, mutant, hydrolase
• 由来する生物種: Rhodococcus sp.
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 61945.08

構造登録者

Rodgers, D.W.,Chow, K.-M.,Fang, L.,Zhan, C.-G. (登録日: 2014-02-20, 公開日: 2014-07-16, 最終更新日: 2024-11-20)

主引用文献

Fang, L.,Chow, K.M.,Hou, S.,Xue, L.,Chen, X.,Rodgers, D.W.,Zheng, F.,Zhan, C.G.
Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.
Acs Chem.Biol., 9:1764-1772, 2014

PubMed Abstract: Cocaine esterase (CocE) is known as the most efficient natural enzyme for cocaine hydrolysis. The major obstacle to the clinical application of wild-type CocE is the thermoinstability with a half-life of only ∼12 min at 37 °C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of ∼6 h at 37 °C is currently in clinical trial Phase II for cocaine overdose treatment. Through molecular modeling and dynamics simulation, we designed and characterized a promising new mutant of E172-173 with extra L196C/I301C mutations (denoted as enzyme E196-301) to produce cross-subunit disulfide bonds that stabilize the dimer structure. The cross-subunit disulfide bonds were confirmed by X-ray diffraction. The designed L196C/I301C mutations have not only considerably extended the in vitro half-life at 37 °C to >100 days, but also significantly improved the catalytic efficiency against cocaine by ∼150%. In addition, the thermostable E196-301 can be PEGylated to significantly prolong the residence time in mice. The PEGylated E196-301 can fully protect mice from a lethal dose of cocaine (180 mg/kg, LD100) for at least 3 days, with an average protection time of ∼94h. This is the longest in vivo protection of mice from the lethal dose of cocaine demonstrated within all studies using an exogenous enzyme reported so far. Hence, E196-301 may be developed to become a more valuable therapeutic enzyme for cocaine abuse treatment, and it demonstrates that a general design strategy and protocol to simultaneously improve both the stability and function are feasible for rational protein drug design.

PubMed: 24919140
DOI: 10.1021/cb500257s

実験手法

X-RAY DIFFRACTION (2.341 Å)