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Structural and Enzymatic Analysis of Orf6, a Novel Dehydratase from a Deep‐Sea Polyunsaturated Fatty Acid Synthase
Author(s) -
RodríguezGuilbe María M,
Schreiter Eric,
Baerga Abel
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb193
Subject(s) - biochemistry , thioesterase , fatty acid synthase , enzyme , coenzyme a , acyl carrier protein , biosynthesis , polyketide synthase , fatty acid , dehydratase , polyketide , polyunsaturated fatty acid , thioester , chemistry , biology , reductase
The polyketide synthase (PKS) genes responsible for eicosapentanoic acid (EPA) biosynthesis in the deep‐sea bacterium, Photobacterium profundum, have been cloned and sequenced, being a total of five polyunsaturated fatty acid (pfa) genes (pfaA, pfaB, pfaC, pfaD and Orf6) enough for EPA production. One of these enzymes is Orf6, a putative thioesterase (TE) domain believed to perform the last step in the fatty acid biosynthesis, catalyzing the hydrolysis and release of the final product from the carrier protein. The purpose of our research is to make a structure‐function analysis of Orf6 TE using its three dimensional structure determined by X‐ray crystallography to guide the re‐engineering of the enzyme. In parallel, we are also performing in vitro enzymatic assays with its likely substrates loaded onto coenzyme A (CoA). The Orf6 structure determined at 1.0 Å resolution reveals a novel hot‐dog fold containing a conserved amino acid, Asp17, in the active site. Results from activity assays indicate that Orf6 does not hydrolyze the CoA thioesters of long‐chain fatty acids. Additional efforts include in vitro assays using CoA thioesters of short‐chain carboxylic acids or acyl carrier protein (ACP) thioesters as substrates for Orf6. Structural and mechanistic knowledge of this seemingly novel enzyme will be important for the design of lipid‐based drugs or the development of industrial applications like biofuels.