Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis
| Autor: | Courtney C. Aldrich, David H. Sherman, Janet L. Smith, William D. Fiers, Greg J. Dodge |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Cyclopropanes
Stereochemistry Protein domain Molecular Conformation Alcohol oxidoreductase Cyanobacteria 010402 general chemistry 01 natural sciences Biochemistry Article Catalysis Synthetic biology Polyketide chemistry.chemical_compound Colloid and Surface Chemistry Bacterial Proteins Protein Domains Biosynthesis Polyketide synthase Dehydration biology 010405 organic chemistry General Chemistry Lyase 0104 chemical sciences Alcohol Oxidoreductases Thiazoles chemistry Dehydratase biology.protein |
| Zdroj: | Journal of the American Chemical Society. 138:16024-16036 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/jacs.6b09748 |
| Popis: | Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all trans trienoate-forming CurK dehydratase, whose kcat of 72 s−1 is more than three-orders of magnitude greater than any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH dehydratases based on incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology. |
| Databáze: | OpenAIRE |
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