Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

Abstract

BackgroundAcyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids.ResultsTo explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family.ConclusionThese acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System

“…It is interesting that the mtFAS system appears to terminate the fatty acid elongation cycle via acyl transferase mechanisms, which is distinct from the ptFAS system that terminates the elongation cycle via a hydrolytic mechanism catalyzed by acyl-ACP thioesterases (Jing et al, 2011). Specifically, mtFAS uses the acyl-transferases AtLpxA, AtLpxD1, and AtLpxD2 (Li et al, 2011) for the assembly of lipid A-like molecules and a combination of lipoyl transferase (Wada et al, 2001) and lipoic acid synthase (Yasuno and Wada, 1998) to generate lipoic acid.…”

Molecular cloning and characterisation of an acyl carrier protein thioesterase gene (CocoFatB1) expressed in the endosperm of coconut (Cocos nucifera) and its heterologous expression in Nicotiana tabacum to engineer the accumulation of different fatty acids

“…Accordingly, CocoFatB1 transcripts are detected not only in the endosperm, but also in the leaves of coconut plants. Thioesterases play a pivotal role in fatty acid synthesis owing to their role in catalysing the terminal reaction of fatty acid biosynthesis, which regulates the fatty acid composition of storage lipids, especially in plant seeds (Brown et al 2010;Jing et al 2011). The expression of thioesterase genes displayed the highest levels in expanding tissues that are typically very active in lipid biosynthesis, such as developing seed endosperm and young expanding leaves (Oo and Stumpf 1979;Sánchez-García et al 2010).…”

“…Because of the potential applications of special seed oils, several studies have focussed on engineering plant thioesterases with medium-chainspecificities. Three acyl-ACP thioesterases (CnFatB1 (JF338903), CnFatB2 (JF338904), CnFatB3 (JF338905)) from coconut have been isolated and characterised (Jing et al 2011). However, their in vivo activities and substrate specificities were only shown in Escherichia coli, with no function analyses performed using plants.…”

“…Fatty acid biosynthesis in higher plants occurs predominantly in the plastids by a de novo iterative 'polymerisation' process, which is commonly primed with the acetyl moiety from acetyl-CoA and proceeds via iterative chain extension through reaction with malonyl-acyl carrier protein (ACP). The synthesis of 16-and 18-carbon (C16 and C18) fatty acids is terminated by the acyl-ACP thioesterase, which catalyses acyl-ACP thioester bond hydrolysis, the terminal reaction of fatty acid biosynthesis that releases a free fatty acid and ACP (Voelker 1996;Jing et al 2011). Therefore, the specificities of thioesterases largely determine the chain lengths of most plant fatty acids (Stumpf 1987).…”

“…This work demonstrated, for the first time, the role of FatB in determing the chain lengths of fatty acids, which spurred efforts to isolate similar enzymes from other plant species with unusual fatty acid phenotypes. Such enzymes included the MCFA-specific thioesterases from Cuphea (Dehesh et al 1996;Leonard et al 1997), American elm (Ulmus americana L.; Voelker et al 1997) and coconut (Jing et al 2011). Because of the potential applications of special seed oils, several studies have focussed on engineering plant thioesterases with medium-chainspecificities.…”