Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities

Abstract

The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

“…Thioesterase activity was assayed in 0.1 ml reactions containing 50 mM Tris-HCl [pH 8.0], 5 mM DTT and [1- 14 C]oleoyl-ACP substrate ranging from 0.02 to 0.08 nmol (30–170 Bq approximately). The oleoyl-ACP was synthetized as described previously ( Aznar-Moreno et al, 2016 ) and the reactions were started by adding of 0.5 μg of the protein preparation ( Serrano-Vega et al, 2005 ). The reactions were carried out at room temperature over 5 min and stopped by adding 0.25 ml of 1 M acetic acid in 2-propanol.…”

“…They are soluble enzymes that display specificity toward short chained acyl-ACPs and have been very important in plant biotechnology ( Eccleston et al, 1996 ; Voelker, 1996 ). By contrast, FatB2 enzymes are widely distributed among plant species, and they display specificity toward palmitoyl- and stearoyl-ACPs when assayed with their physiological acyl-ACP substrates ( Dörmann et al, 2000 ; Salas and Ohlrogge, 2002 ; Aznar-Moreno et al, 2016 ). The role of these enzymes in correct plant development has been demonstrated by reverse genetics ( Bonaventure et al, 2003 ), and they are probably involved in supplying the saturated fatty acids necessary for the synthesis of distinct essential metabolites, such as sphingolipid long chain bases ( Chen et al, 2008 ).…”

“…Based on sequence alignments, these enzymes have been further classified into two sub-families, called FatA and FatB ( Jones et al, 1995 ). The FatA and FatB enzymes differ in their substrate specificity, with FatA showing high substrate specificity for monounsaturated oleoyl-ACP ( Salas and Ohlrogge, 2002 ; Serrano-Vega et al, 2005 ; Sánchez-García et al, 2010 ; Moreno-Pérez et al, 2011 ; Aznar-Moreno et al, 2016 ). Within the FatB subfamily, two groups of enzymes can be found that are referred to as FatB1 and FatB2.…”

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

“…A spinach oleoyl-ACP (FATA-type) thioesterase is known to display strong preference for oleoyl-ACP linked to one plastid-expressed ACP isoform over another (Ohlrogge et al, 1979). In Helianthus annuus (sunflower), the substrate preferences of a seed-expressed FATA and FATB show subtle differences in terms of fatty acyl chain length (16 vs 18) and saturation level preference, depending on which of three plastid ACP isoforms the fatty acyl chain is attached (Aznar-Moreno et al, 2016). Differential expression of various ACP isoforms along with acyl-ACP thioesterases in plants may therefore serve to fine-tune lipid biosynthesis.…”

A high yield optimized method for the production of acylated ACPs enabling the analysis of enzymes involved in P. falciparum fatty acid biosynthesis

“…EcAAS has been shown to accept various types of fatty acids as well as holo-ACPs of different organisms. This method has been applied in combination with saturated fatty acids [10] , [20] , [21] , [22] , [23] , [24] , [25] , but also unsaturated (C16:1 Δ9 , C18:1 Δ9 ) [26] and β-hydroxylated fatty acid chains [27] , [28] . However, the protocols usually include the detergent Triton X-100 to stabilize EcAAS, which renders difficult all downstream assays involving the final acyl-ACP substrates.…”