In vitro analysis of the 6-deoxyerythronolide B synthase : investigating substrate tolerance of the full system and roles of conserved ketosynthase active site residues
Abstract/Contents
- Abstract
- Natural products have been a rich source of pharmaceutically active compounds since before modern medicine. Polyketides are a class of natural products that are biosynthesized by a set of enzymes homologous to the fatty acid synthase. While there are three major classes of polyketide synthases (PKSs) that produce these promising molecules, this thesis will focus on Type I PKSs (also known as assembly line PKSs), which house their enzymatic subdomains within a single polypeptide 'module' and form an assembly line that sequentially processes a small molecule one module at a time. This thesis focuses on a particular Type I PKS, namely the 6-deoxyerythronolide B synthase (DEBS), which produces the macrolactone core of the antibiotic Erythromycin. DEBS has been studied for more than twenty years and serves as a prototypical assembly line PKS with numerous characterizations of both its basic biochemistry as well as implementation of engineering strategies. Using the power of in vitro reconstitution, this thesis investigates a number of interesting properties of the synthase, from its substrate tolerance, to its modular catalytic cycle, to the specific roles of individual amino acids in a subdomain active site. This thesis first describes the complete in vitro reconstitution of DEBS, which allowed for unprecedented control over the system. With this advancement, we were able to probe its substrate tolerance directly, investigating its product profile in the presence of sterically smaller and bulkier extender units. In addition to understanding its natural promiscuity towards non-native extender units, we tested an engineering strategy using a trans-AT to site selectively generate macrolactone analogs. This thesis then examines a remarkable feature of Type I PKSs: their ability to process polyketide chains vectorially (i.e. the growing chain always moves in one direction towards the end of the synthase). We observed mono-occupancy of a PKS module with a growing polyketide chain, despite two available active sites, and refined the catalytic cycle of a PKS module. This newly proposed cycle involves a ketosynthase (KS) whose preference for an acyl-carrier protein is dependent upon the acylation state of its cognate acyl-carrier protein. Specifically, the KS is inaccessible to an upstream acyl-carrier protein after the decarboxylative Claisen condensation within its active site, but becomes accessible again once its cognate ACP has released its newly elongated chain downstream. Finally, it probes more deeply into the mechanism of a KS, examining ten active site residues through the generation of functionally deficient mutants to analyze each residue's role in catalysis. We noted some differences between the KS of a PKS and KSs of previously characterized fatty acid synthases. A few as yet uncharacterized residues may play an important role in the tight regulation of PKS module occupancy discussed above. Broadly, this thesis builds on previous characterization efforts of this remarkable class of natural product producers. With careful analysis of the inner-workings of these incredible machines, the longstanding goal of rationally engineered PKSs is slowly coming into focus. Although still distant from widespread inclusion in drug discovery efforts, engineered PKSs have a promising future.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Robbins, Thomas Frederic |
|---|---|
| Associated with | Stanford University, Department of Chemistry. |
| Primary advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Du Bois, Justin |
| Thesis advisor | Kool, Eric T |
| Advisor | Du Bois, Justin |
| Advisor | Kool, Eric T |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Thomas Frederic Robbins. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Thomas Frederic Robbins
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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