Catalytic mechanism, computational design, and crystal structure of a highly specific and efficient benzoylecgonine hydrolase

Xiabin Chen; Yun Zhang; Junsen Tong; Pengfei Ouyang; Xingyu Deng; Jie Zhang; Huan Liu; Yihui Hu; Weixuan Yao; Jiye Wang; Xia Wang; Shurong Hou; Jianzhuang Yao

doi:10.1016/j.ijbiomac.2024.137767

Catalytic mechanism, computational design, and crystal structure of a highly specific and efficient benzoylecgonine hydrolase

Int J Biol Macromol. 2024 Nov 17:137767. doi: 10.1016/j.ijbiomac.2024.137767. Online ahead of print.

Authors

Xiabin Chen¹, Yun Zhang², Junsen Tong², Pengfei Ouyang², Xingyu Deng², Jie Zhang², Huan Liu², Yihui Hu², Weixuan Yao³, Jiye Wang³, Xia Wang⁴, Shurong Hou⁵, Jianzhuang Yao⁶

Affiliations

¹ School of Pharmacy, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China. Electronic address: xch226@hznu.edu.cn.
² School of Pharmacy, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China.
³ Key Laboratory of Drug Prevention and Control Technology of Zhejiang Province, Zhejiang Police College, Hangzhou, Zhejiang 310053, China.
⁴ School of Biological Science and Technology, University of Jinan, Jinan, Shandong 250022, China.
⁵ School of Pharmacy, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China. Electronic address: houshurong@hznu.edu.cn.
⁶ School of Biological Science and Technology, University of Jinan, Jinan, Shandong 250022, China. Electronic address: bio_yaoj@ujn.edu.cn.

PMID: 39561846
DOI: 10.1016/j.ijbiomac.2024.137767

Abstract

Enzyme therapy for cocaine detoxification should break down both cocaine and its primary toxic metabolite, benzoylecgonine (BZE), which is also the main form of cocaine contaminant in the environment. An ideal BZE-metabolizing enzyme (BZEase) is expected to be highly efficient and selective in BZE hydrolysis. Here, BZEase4 was engineered from bacterial cocaine esterase (CocE) by our reactant state-based enzyme design theories (RED), which has a 34,977-fold improved substrate discrimination between BZE and the neurotransmitter acetylcholine (ACh), compared with wild-type CocE. Under the physiological concentrations of BZE and ACh, the reaction velocity of BZEase4 against BZE is 2.25 × 10⁶-fold higher than it against ACh, suggesting BZEase4 has extremely high substrate selectivity for BZE over ACh to minimize the potential cholinergic side-effects. This study provides additional evidence supporting the further development of BZEase4 toward a promising therapeutic for cocaine overdose, a potentially effective and eco-friendly enzymatic method for BZE degradation in the environment.

Keywords: Acetylcholine; Benzoylecgonine; Cocaine detoxification; RED theory.