title: Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.
abstract: Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.
authors.affiliation: Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1., 41013, Seville, Spain.|Department of Plant Biology and Crop Science, Rothamsted Research, Harpenden, AL5 2JQ, Herts, UK.
authors.forename: Irene
authors.initials: I
authors.lastname: González-Thuillier
authors.affiliation: Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1., 41013, Seville, Spain. mvc@ig.csic.es.
authors.forename: Mónica
authors.initials: M
authors.lastname: Venegas-Calerón
authors.affiliation: Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1., 41013, Seville, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Sánchez
authors.affiliation: Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1., 41013, Seville, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Garcés
authors.affiliation: Biology Department, Copenhagen University, Ole Maaloees Vej 5, 2200, Copenhagen N, Denmark.
authors.forename: Penny
authors.initials: P
authors.lastname: von Wettstein-Knowles
authors.affiliation: Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1., 41013, Seville, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Martínez-Force
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics*; D006368:Helianthus / Q000201:enzymology* / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D010940:Plant Proteins; D064429:Fatty Acid Synthases; D006836:Hydro-Lyases; C036180:beta-hydroxyacyl-(acyl-carrier-protein)dehydrase
keywords: Fatty acid synthase (FAS); Oil biosynthesis; Substrate specificity; Sunflower; β-Hydroxyacyl-[ACP] dehydratase (HAD)