Exploring the programming rules of highly reducing polyketide synthases by analyzing their kinetic parameters
- Author(s): Wang, Bo
- Advisor(s): Tang, Yi
- et al.
Fungal highly reducing polyketide synthases (HR-PKS), which contains three beta-keto processing domains: ketoreductase (KR), dehydratase (DH) and enoylreductase (ER), have higher degree of complexity in their biosynthetic programming rules than that of bacterial PKSs. In order to decipher the relationship between protein sequences and the structures of their products, model fungal HR-PKS LovB and its product lovastatin were studied. In each elongation step, LovB chooses specific combinations of modification domains. This thesis aimed at exploring the programming rules in HR-PKS based on kinetic studies. Specifically, methods for quantifying kinetic parameters of the KR and MT (methyltransferase) domains of HR-PKS were established. Ketoreduction reaction was measured based on NADPH consumption using Plate Reader and methylation reaction was measured according to the desired ions intensity in the positive ionization mode of LC-MS. The fusarielins family was also studied and served as a parallel and complementary system to lovastatin.