Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase
Abstract/Contents
- Abstract
- Modular polyketide synthases (PKSs) are a family of multifunctional catalysts that synthesize a seemingly endless variety of complex natural products, including many molecules that have demonstrated immense therapeutic value in diverse clinical settings. These complex natural products are built from simple coenzyme A-derived precursors in a step-wise fashion, utilizing an assembly line architecture. Understanding the mechanism by which these molecular machines achieve this remarkable feat, with the concomitant goal of rationally reprogramming the assembly line to generate variants of the natural polyketide product with novel properties, has been a longstanding goal of the PKS field. In this assembly line architecture, a set of individual protein domains, known as a module, performs one round of polyketide chain elongation and associated chemical modifications, whereafter the growing chain is translocated to the next PKS module. In particular, every module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze polyketide chain elongation by forming a C-C bond via decarboxylative Claisen condensation. The same ACP then engages the KS domain of the next module to facilitate forward chain translocation of the newly elongated polyketide chain. A lack of understanding of the mechanism for this orderly unidirectional progress of the growing polyketide chain represents a fundamental challenge in the study and rational engineering of assembly line enzymology. In fact, even though KS-ACP interactions have been shown to be specific in both chain elongation and chain translocation, a general model for KS-ACP recognition has proven elusive; it is now apparent that identification of a universal KS-ACP recognition epitope is central to realizing the goal of rational PKS engineering. In light of the above-mentioned challenges, the studies undertaken in this thesis sought to answer three distinct, but related, questions. First, what is the molecular basis of KS-ACP recognition in the context of chain elongation and chain translocation? Second, are these recognition epitopes modular, i.e., can they be predictably interchanged to facilitate communication between heterologous KS-ACP pairs? Third, what insights can a deeper understanding of KS-ACP recognition provide to explain the unidirectional translocation of growing polyketide chains through these enzymatic assembly lines? Using both experimental and computational approaches, we have decoded the molecular basis for KS-ACP recognition in a prototypical PKS, 6-deoxyerythronolide B synthase (DEBS). Surprisingly, KS-ACP recognition is controlled by entirely different regions of the ACP domain during chain elongation versus chain translocation. Significantly, the residues comprising these regions could be predictably interchanged to promote productive KS-ACP interactions in heterologous pairs. Building on these results, we identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along DEBS. As a test of this model, module 3 of DEBS was re-engineered to catalyze two successive rounds of chain elongation as opposed to the single round that is ordinarily catalyzed by PKS modules. Our results indicate that high selectivity has been evolutionarily programmed at two types of KS-ACP protein-protein interfaces, i.e., chain elongation and chain translocation, which are present repetitively along naturally occurring PKS assembly lines, and control the orderly unidirectional progress of the growing polyketide chain. These results offer unprecedented insights into the control of assembly line biosynthesis, and should prove invaluable in our endeavor to harness the programmable biochemistry of these remarkable assembly lines.
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 | Kapur, Shiven |
|---|---|
| Associated with | Stanford University, Department of Chemistry. |
| Primary advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Cegelski, Lynette |
| Thesis advisor | Du Bois, Justin |
| Advisor | Cegelski, Lynette |
| Advisor | Du Bois, Justin |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Shiven Kapur. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Shiven Kapur
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