Insights into the Three-Dimensional Structure of Amorpha-4,11-diene Synthase and Probing of Plasticity Residues.

Abstract

Amorphadiene synthase (ADS) is known for its vital role as a key enzyme in the biosynthesis of the antimalarial drug artemisinin. Despite the vast research targeting this enzyme, an X-ray crystal structure of the enzyme has not yet been reported. In spite of the remarkable difference in product profile among various sesquiterpene synthases, they all share a common α-helical fold with many highly conserved regions especially the bivalent metal ion binding motifs. Hence, to better understand the structural basis of the mechanism of ADS, a reliable 3D homology model representing the conformation of the ADS enzyme and the position of its substrate, farnesyl diphosphate, in the active site was constructed. The model was generated using the reported crystal structure of α-bisabolol synthase mutant, an enzyme with high sequence identity with ADS, as a template. Site-directed mutagenesis was used to probe the active site residues. Seven residues were probed showing their vital role in the ADS mechanism and/or their effect on product profile. The generated variants confirmed the validity of the ADS model. This model will serve as a basis for exploring structure-function relationships of all residues in the active site to obtain further insight into the ADS mechanism.