The role of the iterative polyketide synthase SgcE in biosynthesis of the nine-membered enediyne C-1027

Abstract

Enediyne natural products are potent antibiotics structurally characterized by an enediyne core containing two acetylenic groups conjugated to a double bond in a nine- or ten-membered carbocycle. The biosynthetic gene cluster for biosynthesis of the 9-membered enediyne C-1027 encodes a novel iterative type I polyketide synthase (PKS) SgcE, which is generally believed to initiate the biosynthetic process. However, the exact role of the synthase in the biosynthesis is controversial. It has been reported that in vivo expression and in vitro assay of SgcE generates an all-trans 1, 3, 5, 7, 9, 11, 13-pentadecaheptaene product, which is proposed to be an octaketide precursor of the nine-membered enediyne core of C-1027. These previous studies also indicated that the activity of the SgcE is strictly dependent on hot dog thioesterase SgcE10. In this thesis, we reconstituted the activity of recombinant SgcE and found that the multifunctional enzyme exclusively produced a new nonaketide product, which was not affected by the thioesterase SgcE10 at low concentrations. In the presence SgcE10 at high concentrations, the polyketide synthase produced a reduced level of the newly identified nonaketide and two additional polyene products, of which one is the precursor to the nonaketide product and the other is the previously identified octaketide molecule (trans-1, 3, 5, 7, 9, 11, 13-pentadecaheptaene). These findings strongly suggests a new probable biosynthetic route to nine-membered endiyne core of C-1027, in which the polyketide synthase SgcE produces the nonaketide as a precursor and the thioesterase SgcE10 serves as a type II thioesterase proofreading the modular synthesis of the nonaketide product. Next, we characterized the polyketide synthase activities by determining the steady-state kinetics of the synthesis of the polyene products and evaluating the synthase’s side activities. In addition, we investigated the acyl carrier protein (ACP) and phosphopantetheinyl transferase (PPTase) domains that are uniquely present in a single polypeptide chain in SgcE. Characterization of the two domains as stand-alone proteins and as integrated domains on SgcE indicates that the flanking sequence of the ACP domain is essential for recognition by the PPTase and that the PPTase domain has high specificity towards its cognate ACP as an integrated domain of the synthase; meanwhile, both the ACP and PPTase domains of SgcE rely on their surrounding sequences for correct folding into an active conformation. These results help us to better understand the important role of the iterative polyketide synthase in the biosynthesis of the nine-membered cores of enediyne natural products.

Date of Award	2011
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology