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

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dc.identifier.uri http://dx.doi.org/10.15488/4303
dc.identifier.uri https://www.repo.uni-hannover.de/handle/123456789/4337
dc.contributor.author Yao, Hao ger
dc.date.accessioned 2019-01-11T09:49:02Z
dc.date.available 2019-01-11T09:49:02Z
dc.date.issued 2019
dc.identifier.citation Yao, Hao: In Vitro investigation of multi-domain fragments of squalestatin tetraketide synthase. Hannover : Gottfried Wilhelm Leibniz Universität, Diss., 2019, 200 S. https://doi.org/10.15488/4303 ger
dc.description.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). ger
dc.language.iso eng ger
dc.publisher Hannover : Institutionelles Repositorium der Leibniz Universität Hannover
dc.rights CC BY 3.0 DE ger
dc.rights.uri http://creativecommons.org/licenses/by/3.0/de/ ger
dc.subject squalestatin tetraketide synthase eng
dc.subject multi-domain fragments eng
dc.subject stereoselectivity eng
dc.subject Squalestatin Tetraketid Synthase ger
dc.subject Multidomän-Fragmente ger
dc.subject in vitro ger
dc.subject Stereoselektivität ger
dc.subject.ddc 540 | Chemie ger
dc.title In Vitro investigation of multi-domain fragments of squalestatin tetraketide synthase ger
dc.type doctoralThesis ger
dc.type Text ger
dc.description.version publishedVersion ger
tib.accessRights frei zug�nglich ger


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