Computational design of a thermostable mutant of cocaine esterase via molecular dynamics simulations

Abstract

Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ∼11 min at physiological temperature (37 °C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the root-mean square deviation and root-mean square fluctuation of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modelling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37 °C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability relationship may be considered as a valuable tool for the computational design of thermostable mutants of an enzyme.

Fig. 1

(A) Time-dependence of the root-mean square deviation (RMSD) of the high-temperature (T = 400 K) MD-simulated atomic positions of all heavy atoms of the catalytic residues (Y44, S117, Y118, D259, and H287) from those in the starting structure. (B) Energy-minimized structure of the T172R/G173Q mutant. The enzyme is represented as colored ribbon. Amino acid residues including the catalytic triad (S117-H287-D259) are shown as stick and colored by atom types. The hydrogen bonding and cation-π interactions are represented as dashed lines with distances labeled. (C) Energy-minimized structure of the L169K mutant, which is shown in a similar way as that in (B). (D) Time-dependence of important internuclear distances from the high-temperature (T = 400 K) MD-simulated L169K mutant. Y44HH-W166NE represents the distance between the hydroxyl hydrogen (HH) of Y44 side chain and the nitrogen (NE) atom of W166 side chain, K169NZ-Y44 stands for the cation-π distance between the nitrogen (NZ) atom on the cationic head of K169 side chain and the center of the phenyl ring of Y44 side chain, and K169HZ-Y44OH refers to the shortest distance between the hydroxyl oxygen of Y44 side chain and the protons on the cationic head of K169 side chain. All of the data are based on the production MD.