Orf6: Biochemical Characterization of a Putative Thioesterase from a Polyunsaturated Fatty Acid Synthase

Abstract

Polyunsaturated fatty acids are made in deep‐sea organisms by the activity of a polyketide synthase multienzyme. The final step in the biosynthesis of polyketides and fatty acids is catalyzed by the activity of the thioesterase (TE) domain, which cleaves the final product off of the carrier protein. However, no TE domain has been identified in any of the known PUFA synthase clusters. We propose that orf6 gene is a candidate TE, since it is conserved among other bacteria with PUFA synthase clusters and is adjacent to the PUFA synthase cluster in Photobacterium profundum. We have cloned, expressed and purified Orf6 protein with the aim of characterizing the protein structurally and functionally. The Orf6 structure, determined by X‐ray crystallography at 1.0Å resolution, demonstrates a hot‐dog fold similar to the 4‐hydroxybenzoyl‐CoA TE family. Results from enzyme assays show that Orf6 has no activity toward long‐chain fatty acids, including the expected full length product eicosapentaenoic acid attached to a CoA carrier. Additional efforts include in vitro assays using CoA thioesters of short‐chain carboxylic acids as well as aromatic carboxylic acids. Structural and mechanistic knowledge of this enzyme will be important for the development of the PUFA synthases as a platform for the production of fatty acids. This work was funded by grant CHE0953254 from the NSF and MBRS‐RISE Program (R25GM061838) of the UPR‐MSC.