A study of the functional domains of the type I iterative polyketide synthase CalE8 in calicheamicin biosynthesis

• Main Author: Murugan, Elavazhagan
• Other Authors: Liang Zhao-Xun
• Format: Theses and Dissertations
• Language: English
• Published: 2012
• Subjects: DRNTU::Science::Biological sciences::Biochemistry
• Online Access: https://hdl.handle.net/10356/50212
• Institution: Nanyang Technological University

Naturally occurring enediynes are potent antibiotics produced by soil and marine microorganisms. Their robust antitumor activities and unique mode of action make them a significant topic of study. The synthesis of the enediyne products is initiated by a type I iterative polyketide synthase (PKS). In this project, we examine the structures and functions of the domains of CalE8, to demarcate the domains of the the iterative PKS from the biosynthetic pathway of the 10-membered enediyne calicheamicin in Micromonospora echinospora spp. The 212 KDa CalE8 contains several domains including the predicted ketoacyl synthase (KS), acyl transferase (AT), ketoreductase (KR) and dehydratase (DH) domains. In addition, CalE8 also contains a postulated acyl carrier protein (ACP) domain and a C-terminal domain with unknown function. The first step of enediyne biosynthesis involves a post-translational modification of the ACP domain by 4’- phosphopantetheinylation. The ACP domain and C-terminal portion of CalE8 were first cloned and expressed as stand-alone proteins to study their functions. The identity of the ACP domain was established by in vitro phosphopantetheinylation using the surfactin PPTase (Sfp) from Bacillus subtilis. The NMR solution of the ACP domain was solved to show that the ACP exhibits some rather distinct structure feature from other ACPs. Furthermore, we found that the C-terminal domain exhibits PPTase activity towards various carrier proteins. Sequence analysis and modeling studies suggest the C-terminal domain is an unusual Sfp-like PPTase domain integrated into CalE8. Finally, the AT, KS, DH and KR domains of the CalE8 PKS were examined thoroughly by bioinformatics tools such as structural modeling to define the domain boundaries and catalytic residues. The individual domains were cloned and expressed in E. coli for structure determination. Although the KS, DH and KR domain proteins were found to be insoluble, the AT domain was soluble and purified for further studies. The access to the soluble AT domain will be valuable for studying the substrate specificity of the AT domain in accepting malonyl-CoA, but not acetyl-CoA as substrate. For characterizing the covalently-attached products of CalE8, an ACP phosphodiesterase capable of cleaving the growing polyketides was examined. This family of phosphodiesterases was found to be highly unstable with the propensity to form precipitate in solution. We have identified a phosphodiesterase (PaAcpH) from Pseudomonas aeruginosa that can be expressed and purified as a soluble protein. The function and substrate specificity of PaAcpH was first validated and examined with several carrier proteins from different pathways. We demonstrate that PaAcpH is indeed able to catalyze the removal of the phosphopantetheinyl moiety and the tethered-intermediates from CalE8. The capability of releasing the polyketide intermediates by PaAcpH is valuable in the study of PKS mechanisms.