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Functional architectures of polyketide synthases

Herbst, Dominik Alexander. Functional architectures of polyketide synthases. 2017, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12567

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Abstract

Microbial polyketide synthases (PKS) are biological factories for the production of potent natural products, which include clinically relevant antibiotics, anti-cancer drugs, statins and more. The exceptional chemical diversity generated by PKSs is encoded in a modular architecture for precursor extension. The domains required for one step of precursor elongation and modification are combined into a functional polypeptide module, which is segregated into a mandatory condensing region for elongation and an optional and variable part for intermediate modification. PKS modules contain integral acyl carrier protein (ACP) domains, flanked by flexible peptide regions. ACPs are used to load substrates and to tether intermediates throughout ongoing synthesis, by linking them as thioesters to a covalently attached phosphopantetheine cofactor. PKS modules can either act iteratively (iPKS) or in a linearly organized assembly line of multiple modules (modPKS), where the nascent polyketide is handed over from one to the next module. The collinearity between synthesis and protein sequence in modPKS holds promise for rational re-engineering in order to produce novel bioactive compounds. Despite their cyclic mode of action, iPKS may employ specific reaction programs, which introduces different substitutions in each iteration by selective use of individual catalytic domains.
At the beginning of the thesis, the architecture of PKS modules as a basis for their modular organization and programmed biosynthesis was unknown. This thesis was focused on structural studies of the architecture of PKS modules, intramodular crosstalk and functional programming. Chapter one provides a comprehensive introduction into the molecular biology of PKS function.
Chapter two provides a hybrid crystallographic model of an iPKS module and demonstrates its relevance also for modPKS. Overlapping crystal structures of a condensing and a complete modifying region provided the first atomic model of a PKS module with a total of 10 catalytic domains. Multiple crystallogrpahically independent copies observed in the 3.75 Å structure of the dimeric modifying region provided snapshots of a variable linker-based architecture with implications for PKS evolution and conformational coupling of reaction steps in the dimeric synthase. Comparative small angle X-ray scattering demonstrates that the iPKS architecture is also representative for tested modPKSs.
Chapter three reports the crystal structure of a programming C-methyltransferase (CMeT) domain at 1.65 Å resolution. The structure reveals a novel N-terminal fold and a substrate binding cavity that accommodates intermediates of various length during iterative biosynthesis. Structural and phylogenetic analysis demonstrates conservation of CMeT domains in PKS as well as homology to an inactive pseudo-CMeT (ΨCMeT) remnant in mammalian fatty acid synthase (mFAS). The data suggest an involvement of the core elongating ketosynthase (KS) domain in PKS programming.
Chapter four provides a visualization of substrate loading in iPKS. A 2.8 Å resolution crystal structure provided detailed insights into an intertwined linker-mediated integration of substrate-loading starter-unit acyltransferase (SAT) domains into an iPKS condensing region. The post-loading state was trapped by mechanism-based crosslinking. Visualization by cryo electron microscopy at 7.1 Å resolution revealed asymmetry of ACP-KS interactions and depicts conformational coupling across the dimeric PKS for coordinated synthesis.
Chapter five integrates the results into the current structural and biological context and discusses current opinions and future perspectives in the field. The results of this thesis reflect the relevance of linker-based connections rather than stable domain-domain interfaces for PKS architecture. This work also highlights mechanisms for conformational coupling for synthesis and substrate channeling in dimeric, but asymmetric, PKS. These insights will support re-engineering iPKS and modPKS assembly lines for the production of novel bioactive compounds, in particular for drug discovery.
Advisors:Maier, Timm and Stahlberg, Henning
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Maier)
UniBasel Contributors:Herbst, Dominik Alexander and Maier, Timm and Stahlberg, Henning
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12567
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (155 Seiten)
Language:English
Identification Number:
Last Modified:18 Apr 2018 13:45
Deposited On:16 Apr 2018 13:55

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