4P08

Engineered thermostable dimeric cocaine esterase

Summary for 4P08

• Entry DOI: 10.2210/pdb4p08/pdb
• Descriptor: Cocaine esterase (2 entities in total)
• Functional Keywords: esterase, disulfide-linked dimer, cocaine, mutant, hydrolase
• Biological source: Rhodococcus sp.
• Total number of polymer chains: 1
• Total formula weight: 61945.08

Authors

Rodgers, D.W.,Chow, K.-M.,Fang, L.,Zhan, C.-G. (deposition date: 2014-02-20, release date: 2014-07-16, Last modification date: 2023-12-27)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (2.341 Å)