title: Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids.
abstract: In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg(2+) to in vitro assays facilitates appearance of radiolabel from (14)C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase.
authors.affiliation: Martek Biosciences Boulder Corporation, Boulder, CO 80301, USA. jmetz@martek.com
authors.forename: James G
authors.initials: JG
authors.lastname: Metz
authors.forename: Jerry
authors.initials: J
authors.lastname: Kuner
authors.forename: Bradley
authors.initials: B
authors.lastname: Rosenzweig
authors.forename: James C
authors.initials: JC
authors.lastname: Lippmeier
authors.forename: Paul
authors.initials: P
authors.lastname: Roessler
authors.forename: Ross
authors.initials: R
authors.lastname: Zirkle
mesh_terms: D000106:Acetate-CoA Ligase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D004281:Docosahexaenoic Acids / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000096:biosynthesis*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D009868:Oomycetes / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D010938:Plant Oils / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000378:metabolism*; D014280:Triglycerides / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D005230:Fatty Acids, Nonesterified; D005231:Fatty Acids, Unsaturated; D010938:Plant Oils; D014280:Triglycerides; D004281:Docosahexaenoic Acids; D008316:Malonyl Coenzyme A; D000255:Adenosine Triphosphate; D013869:Thiolester Hydrolases; D054889:Fatty Acid Synthase, Type II; D000106:Acetate-CoA Ligase; D008274:Magnesium; C026219:docosapentaenoic acid