title: Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.
abstract: Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to β-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.
authors.affiliation: Department of Biochemistry, University of Puerto Rico School of Medicine, San Juan, 00936-5067, Puerto Rico.
authors.forename: Delise
authors.initials: D
authors.lastname: Oyola-Robles
authors.forename: Darren C
authors.initials: DC
authors.lastname: Gay
authors.forename: Uldaeliz
authors.initials: U
authors.lastname: Trujillo
authors.forename: John M
authors.initials: JM
authors.lastname: Sánchez-Parés
authors.forename: Mei-Ling
authors.initials: ML
authors.lastname: Bermúdez
authors.forename: Mónica
authors.initials: M
authors.lastname: Rivera-Díaz
authors.forename: Néstor M
authors.initials: NM
authors.lastname: Carballeira
authors.forename: Abel
authors.initials: A
authors.lastname: Baerga-Ortiz
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D064429:Fatty Acid Synthases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; D064429:Fatty Acid Synthases; D006836:Hydro-Lyases