Sunday, July 24, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
Polyunsaturated fatty acids (PUFAs) 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 typically 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 the orf6 gene is a plausible 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 TE with the aim of characterizing the protein both structurally and functionally in order to elucidate its role as a thioesterase. The Orf6 structure was determined at 1.0 Å resolution and it demonstrates a hot-dog fold with a conserved amino acid, Asp17, in the enzyme’s active site. Results from enzyme assays indicate that Orf6 shows no activity toward the expected full length product eicosapentanoic acid (EPA) attached to a CoA carrier. However, Orf6 was seen to be active towards long-chain saturated fatty acids. 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.