The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase

Abstract

Background: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization.

Results: To create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cbeta of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50 degrees C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies.

Conclusion: Addition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation.

Figure 1

Arrhenius plots of thermal inactivation rate constants of mutated DmAChE (in red) compared to wild type (in blue). k: denaturation first order rate constant (in min^-1).

Figure 2

Effect of mutations on acetylthiocholine hydrolysis versus substrate concentration (log scale). (blue dots): wild type; (red dots): mutant, Acetylthiocholine concentration in micromoles per liter; v/[Et] specific activity in s^-1.

Figure 3

Position of mutation m2 (I327/D375). The cross link has been colored in red. The disulfide bridge links two subdomains of the protein.