Expression, Purification and Preliminary Activity of a Novel Dehydratase Domain from a Polyketide Synthase.

Abstract

Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and important components of human health. PUFAs from deep‐sea bacteria are synthesized by a modular polyketide synthase consisting of several domains including two dehydratase (DH) domains, DH1 and DH2, which are responsible for the introduction of cis or trans double bonds into the final product. In order to study the mechanism and substrate specificity of these DH domains, protein fragments were expressed containing one or both domains. Domain boundaries were chosen by bioinformatic analysis using the Udwary‐Merski Algorithm (UMA) for the prediction of protein linkers. Recombinant proteins were purified by nickel agarose and anion exchange chromatography. Enzyme assays were performed by monitoring the consumption of synthetic substrates by UV spectroscopy. Results from the UMA sequence analysis suggest the presence of two additional, unexpected domains preceding DH1 and DH2. Results suggest that these unexpected domains are important for protein structure and stability, since constructs that lack these domains are insoluble. Preliminary enzyme assays show that all soluble DH‐containing constructs were inactive towards the short‐chain thioester substrate mimic, suggesting that longer thioesters substrate mimics will be required in order to assay dehydratase activities.