Structural and functional studies of enzymes from the enediyne polyketide biosynthetic pathway
Liew, Chong Wai
Date of Issue2013
School of Biological Sciences
Enediyne polyketides are extremely potent antitumor antibiotics with a remarkable core structure consisting of two acetylenic group conjugated to a double bond within either 9-membered ring (bicycle-[7.3.0]-dodecadienediyne) or 10-membered ring (bicycle-[7.3.1]-tridecadiynene). Despite their structural differences, all enediyne antibiotics damage DNA via a rapid enediyne cycloaromatization to form highly reactive diradical species capable of inducing oxidative DNA strand scission. Due to their astonishing capacity in cleaving DNA, some of the enediynes have been conjugated to tumor specific monoclonal antibiotic. Besides, the application of polymer assisted delivery devices have also led to the clinical success of enediyne. With the sequencing of gene clusters responsible for enediyne production in 2002, studies on the biosynthesis of enediynes finally have ushered in a new era. The identification of the “minimal enediyne cassette” in the gene cluster also led the way for scientists to look into the biosynthetic pathway of the enediyne core. With the genomic information in hand, we could set out to explore the biosynthetic origin of the enediyne core of the enediyne polyketide synthase (PKSE). After successfully cloning, expressing and purifying the three proteins encoded by the genes from the minimal cassette, PKSE (CalE8, SgcE, and DynE8), TEBC (CalE7, SgcE10 and DynE7) and UNBL (CalU15, SgcE3 and DynU15), in E. coli expression system, a series of biochemical and structural studies have been carrier out to examine the structure and function of the proteins. The roles of these proteins in the early steps of enediyne core biosynthetic pathway were investigated. Various products of CalE8, SgcE and DynE8 have been synthesized and identified through in vitro biochemical assays. The crystal structure of the acytransferase domain of DynE8, as well as its binary complexes with glycerol, acetate and malonate, was determined to yield insight into the structure, and function of the substrate-transferring domain. Moreover, the crystal structure of thioesterases (CalE7, SgcE10 and DynE7) and ligand-enzyme complex were determined that together with mutagenesis studies reveal a remarkable induced-fit mechanism during substrate binding and a novel catalytic mechanism in releasing the polyketide product.