A hot-dog fold thioesterase is found in all known biosynthetic gene clusters of enediyne natural products. Previous studies have suggested that it is a type I thioesterase responsible for releasing the polyketide product from the iterative type I polyketide synthease (PKS). However, studies from our research group shown that the enediyne PKS from the nine-membered enediyne C-1027 cluster releases its product autonomously and the associated thioesterase SgcE10 is likely a type II thioesterase proofreading the polyketide synthesis. In this thesis, SgcE10 is characterized, and compared to CalE7, which has been structurally and mechanistically as a type I thioesterase in the ten-membered enediyne core of calicheamicin. It is found to exhibit high activity towards the acyl-CoA substrates wit...[ Read more ]

A hot-dog fold thioesterase is found in all known biosynthetic gene clusters of enediyne natural products. Previous studies have suggested that it is a type I thioesterase responsible for releasing the polyketide product from the iterative type I polyketide synthease (PKS). However, studies from our research group shown that the enediyne PKS from the nine-membered enediyne C-1027 cluster releases its product autonomously and the associated thioesterase SgcE10 is likely a type II thioesterase proofreading the polyketide synthesis. In this thesis, SgcE10 is characterized, and compared to CalE7, which has been structurally and mechanistically as a type I thioesterase in the ten-membered enediyne core of calicheamicin. It is found to exhibit high activity towards the acyl-CoA substrates with long chain acyl group. By site-directed mutagenesis and kinetic analysis, we found that the conserved Arg39 and other active-site residues such as Asn21, Asn25 and Tyr31 are essential to the hydrolytic activity of SgcE10. Mutation of these conserved residues completely abolishes the enzymatic activity. Interestingly, most of these results are consistent with those obtained for CalE7 characterized as a type I thioesterase, suggesting that these thioesterases may assume an identical catalytic role in the intriguing biosynthetic steps leading to the enediyne cores.