In Vitro investigation of multi-domain fragments of squalestatin tetraketide synthase

Abstract

Squalestatin tetraketide synthase (SQTKS) is a fungal iterative highly-reducing polyketide synthase (HR-PKS) that catalyzes the biosynthesis of the tetraketide side chain of squalestatin-S1 which is a potent squalene synthase inhibitor and can be potentially used to treat serum cholesterol related diseases. The SQTKS protein is one of the simplest iterative type I HR-PKS as all the iterative β-modification domains are present in an active state and in which a degree of programming occurs. To investigate the programming of the HR-PKS, detailed in vitro and stereochemical studies are fundamental. In this thesis, we have cloned and overexpressed several large multi-domain fragments of SQTKS in E. coli. These fragments have been isolated under a rational purification design and analyzed biochemically and biophysically. Using pantetheine substrates (analogs of the true ACP-bound substrate in SQTKS), kinetic studies of enoyl reduction show that the ER of DH-KR tetradomain is capable of transforming various substrates and it performs the programming function by its inability to reduce the final tetraketide substrate. These results are in accord with the kinetic studies of the isolated SQTKS ER monodomain. As an effect of domain-domain interaction, different stereoselectivities in enoyl reduction have been found by in vitro assays with the SQTKS ER monodomain and with a multidomain construct. With the tetradomain protein, the stereopreference of keto reduction has been studied. From the comparison between the mammalian fatty acid synthase (mFAS) and the SQTKS in stereoselectivity, they are identical, which reinforces the idea that the HR-PKS and mFAS evolved from a common ancestor. To investigate the methyltranfer mechanism, in vitro assays with isolated SQTKS C- methyl transferase (CMeT) monodomain have been studied. In addition, the effectiveness of using the pantetheine substrate as analogs of the original ACP-bound substrate was investigated in this research. These studies indicate that the full methyltransfer activity of SQTKS CMeT may need the cooperation of the acyl carrier protein (ACP).