Cloning, characterization and structural model of a FatA-type thioesterase from sunflower seeds (Helianthus annuus L.)

Abstract

The substrate specificity of acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) determines the fatty acids available for the biosynthesis of storage and membrane lipids in seeds. In order to determine the mechanisms involved in the biosynthesis of fatty acids in sunflower seeds (Helianthus annuus L.), we isolated, cloned and sequenced a cDNA clone of acyl-ACP thioesterase from developing sunflower seeds, HaFatA1. Through the heterologous expression of HaFatA1 in Escherichia coli we have purified and characterized this enzyme, showing that sunflower HaFatA1 cDNA encodes a functional thioesterase with preference for monounsaturated acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/K(m)) in catalyzing oleoyl-ACP, both in vivo and in vitro. By comparing this sequence with those obtained from public databases, we constructed a phylogenetic tree that included FatA and FatB thioesterases, as well as related prokaryotic proteins. The phylogenetic relationships support the endosymbiotic theory of the origin of eukaryotic cells and the suggestion that eubacteria from the delta-subdivision were the guest cells in the symbiosis with archaea. These prokaryotic proteins are more homologous to plant FatB, suggesting that the ancient thioesterases were more similar to FatB. Finally, using the available structure prediction methods, a 3D model of plant acyl-ACP thioesterases is proposed that reflects the combined data from direct mutagenesis and chimera studies. In addition, the model was tested by mutating the residues proposed to interact with the ACP protein in the FatA thioesterase by site-directed mutagenesis. The results indicate that this region is involved in the stabilization of the substrate at the active site.

Effect of a mutagenized acyl-ACP thioesterase FATA allele from sunflower with improved activity in tobacco leaves and Arabidopsis seeds

“…This model was consistent with previous research on the specificity of these enzymes, which were altered by changes in different protein residues and domain swapping (Yuan et al 1995(Yuan et al , 1996Salas and Ohlrogge 2002). It is hypothesized that the active site of these enzymes displays a hot-dog fold pattern, with a catalytic triad of amino acids similar to that in papain (Mayer and Shanklin 2005;Serrano-Vega et al 2005). Furthermore, the regions interacting with the substrate were identified, including a hydrophobic pocket that was a clear candidate in determining substrate specificity (Serrano-Vega et al 2005;Mayer and Shanklin 2007).…”

“…Sunflower acyl-ACP thioesterase cDNA (HaFatA) has been previously cloned and characterized and is probably the dominant form measurable in crude extracts from sunflower seeds (Serrano-Vega et al 2005). Using the acyl-ACP thioesterase structural models that have been previously developed (Mayer and Shanklin 2005;Serrano-Vega et al 2005) and bioinformatic tools was possible to identify amino acid residues candidates to be involved in substrate specificity of these proteins.…”

“…Using the acyl-ACP thioesterase structural models that have been previously developed (Mayer and Shanklin 2005;Serrano-Vega et al 2005) and bioinformatic tools was possible to identify amino acid residues candidates to be involved in substrate specificity of these proteins. In the case of…”

“…FatA are dimeric enzymes, encoded by nuclear genes, which are posttranslationally imported into plastids, involving the removal of an N-terminal transit peptide. HaFatA is expressed at high levels in sunflower seeds and has higher catalytic efficiencies than other FatAs from plant species previously described (Serrano-Vega et al 2005). Alterations in the binding pocket were affected by the exchange of selected amino acid residues with tryptophan which is hypothesized to interfere with pocket size …”

“…It is hypothesized that the active site of these enzymes displays a hot-dog fold pattern, with a catalytic triad of amino acids similar to that in papain (Mayer and Shanklin 2005;Serrano-Vega et al 2005). Furthermore, the regions interacting with the substrate were identified, including a hydrophobic pocket that was a clear candidate in determining substrate specificity (Serrano-Vega et al 2005;Mayer and Shanklin 2007).…”