ACP-thioesterase

It is worth mentioning that metabolic engineering of E. coli recently provided recombinant strains which synthesized PHAMCL from gluconate. For this, beside phaC2Po or phaClPa> the thioesterase I from E. coli (TesA) [128] or the acyl-ACP thioesterase from Umbellularia californica [129], respectively, were expressed in E. coli. However, the amounts of PHAMCL accumulated in the cells were rather low, and this artificial pathway was not very efficient. 

Fig. 4. Modification of plant metabolic pathways for the synthesis of poly(3HAMCL) in peroxisomes. The pathways created or enhanced by the expression of transgenes (P. aeruginosa PHA synthase and C. lanceolata decanoyl-ACP thioesterase) and of mutant alleles of plant fatty acid desaturase genes are highlighted by bold arrows and the enzymes involved underlined...

Modulation of the quantity and/or quality of poly(3HAMCL) synthesized in peroxisomes was also achieved by modifying the endogenous fatty acid biosynthetic pathway [58]. For example, expression of the peroxisomal PHA synthase in an A. thaliana mutant deficient in the synthesis of triunsaturated fatty acids [59] resulted in the synthesis of a PHA having an almost complete absence of triunsaturated 3-hydroxyacid monomers [58]. In a different strategy, expression of a fatty acyl-ACP thio esterase in the plastid was combined with the expression of a peroxisomal PHA synthase [58]. Fatty acyl-ACP thioesterases are... 

Fig. 2. Predicted domain organisation of the DEBS Proteins. Ketoacyl Synthase (KS) Acyl Transferase (AT) Dehydratase (DH) Enoyl Reductase (ER) Keto Reductase (KR) Acyl Carrier Protein (ACP) Thioesterase (TE). Each domain is represented by a box with coded shading whose length is proportional to the size of the domain (KR) indicates an inactive KR domain. The ruler indicates the residue number within the primary structure of the constituent proteins. Linker regions are shown in proportion...

The AT homologue ZhuC in the R1128 PKS was found to be an acetyl-ACP thioesterase that is required to suppress acetate priming. In vitro PKS reconstitution experiments with ZhuC showed that its addition to the act and tcm minimal PKSs significantly attenuated the synthesis of acetate-primed polyketides (30). ZhuC was shown to catalyze the rapid hydrolysis of acetyl-ACP to yield acetate and holo-ACP (kct >150 min ). In contrast, ZhuC does... 

Figure 3. Priming and editing mechanisms for the RI128 initiation module. The acyl group carried by ZhuG is shuttled to the KS-CLF of the minimal PKS. The acyl-primed KS-CLF is then able to elongate the starter unit into a full length polyketide. ZhuC serves as an acetyl-ACP thioesterase.

Acyl-CoA thioesterase enzymes (EC 3.1.2.-), although their catalytic activity simply entails the hydrolysis of CoA and ACP thioesters to release the fatty acids and other carboxylic acids bound to them (Equation (19)), have wide and varied physiological functions that includes the regulation of fatty acid metabolism and playing a central role in the biosynthesis of polyketide and nonribosomal peptide-based metabolites (especially the macrocyclic versions) and the degradation of aromatic compounds. These enzymes are thoroughly discussed in several recent reviews as well as the relevant chapters of this series that include fatty acids, polyketides, and nonribosomal peptide biosynthesis ° ° (see Chapters 1.05,1.02, and 5.19) therefore, only a brief overview of the structural and mechanistic diversity of acyl-CoA and acyl-ACP thioesterases is provided in this section. 

Acyl-ACP thioesterases terminate fatty acid synthesis. 103... 

Laurie acid production Acyl-ACP thioesterase California Bay Rapeseed... 

One of the major non-food uses of vegetable oils (approximately 5(X) million pounds of oil per annum in the US) is the production of soaps, detergents, and other surfactants. The solubility and other physical properties of medium-chain fatty acids and their derivatives make them especially suited for surfactant manufacture. Coconut and palm kernel oils, which contain 40-60% lauric acid (12 0), are current major feedstocks for the surfactant industry. The mechanism of synthesis of lauric and other medium-chain fatty acids in plants involves the action of a medium-chain acyl-ACP thioesterase which terminates fatty acid synthesis after a 10 or 12 carbon chain has been assembled (M. Pollard,... 

Fig. 11. Genetic engineering of rapeseed oil. A high level of lauric acid was achieved by expressing a medium-chain acyl-ACP thioesterase (MCTE) from California Bay in the transgenic seeds. This enzyme intercepts the fatty acid synthesis pathway at 12 carbons and hydrolyzes the fatty acid from its ACP carrier. MoI% of major fatty acids in a typical canola cultivar are compared to the composition achieved through genetic engineering.

Jones, A., Davies, H.M., Voelker, T.A. 1995. Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases. Plant Cell 7 359-371. 

The three main products formed during the FA synthesis in the stroma of the plastid are hydrolyzed by two possible acyl-ACP thioesterases (FAT) FAT A and FAT B. FAT A has preference for oleoyl-ACP but also hydrolyzes stearoyl- and palmitoyl-ACP. FAT B, which has preference for saturated FA-ACP and especially palmitoyl-ACP, can also hydrolyze oleoyl-ACP (Voelker Kinney, 2001). 

Li, J., Szittner, R. and Meighen, E. A. 1998, Tryptophan Fluorescence of the lux-Specific Vibrio harveyi Acyl-ACP Thioesterase and Its Tryptophan Mutants Structural Properties and Ligand-Induced Conformational Change. Biochemistry 37, 16130 -16138. 

The modification of vegetable oil composition involves, on the one hand, a reduction in the saturated FA concentration. This was achieved by DuPont, which reduced the concentration of these acids in soybean oil from 15% to less than 4% by suppressing the activity of a type 11 acetyl-ACP thioesterase (Kinney, 1996). The other direction of genetic modifications leads to an increase in the concentration of the unsaturated FA in oils. For example, greater unsaturated FA concentrations in canola seed oil (from 68% to 83%) were obtained by suppressing the activity of the... 

EN] BRASSICA PLANT COMPRISING MUTANT FATTY ACYL-ACP THIOESTERASE ALLELES. .. 

FatA. and FatB - acyl-ACP thioesterases hydrolyzing predominantly oleoyl-ACP and diverse saturated and unsaturated acyl ACP, respectively ENR - enoyl ACP reductase KAR - ketoacyl ACP reductase KAS - ketoacyl synthase MCMT - malonyl-CoA ACP malonyl transferase HADH -hydroxyacyl ACP dehydratase. 

The termination of elongation is catalyzed by ACP-thioesterases (enzymes belonging to the class of acyl-ACP hydrolases). These enzymes hydrolyze acyl-ACP with the formation of free FA, whieh ean eross the plastid membrane to be reactivated outside the organelle [11]. 

Enzymes of the prokaryotic pathway are localized in plastids, whereas enzymes of the eukaryotic pathway - in the cytosol and ER. In the prokaryotic pathway, FA acyls are directly transferred from AGP to G3P, whereas in the eukaryotic pathway, FA are separated from ACP by acyl-ACP thioesterases and released free FA then are transported in the cytoplasm, where they are converted into acyl-CoA. During the synthesis of membrane and storage lipids acyl groups are used in the ER by acyltransferases of the eukaryotic G3P pathway [66]. In dependence on subcellular localization, these enzymes may differ in their structure, thus forming independent clusters in phylogenetic investigations [67]. 

Weselake, RJ Taylor, DC Rahman, MH Shah, S Laroche, A MeVetty, P Harwood, J. Increasing the flow of carbon into seed oil. Biotechnol Adv., 2009,27, 866-78. Pollard, MR Anderson, L Fan, C Hawkins, D Davies, H. A speeific a( l-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica. Arch. Biochem. Biophys., 1991, 284, 306-312. 

Salas, JJ Ohlrogge, JB. Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases. Biochem. Biophys., 2002,403, 25-34. 

These chain lengths may be maintained by the combined action of the three keto-acyl synthase enzymes (KAS I-III), and relatively specific thioesterases that convert acyl-ACP molecules to the corresponding CoA derivatives. Most plants have an acyl-ACP thioesterase with the highest activity for oleyl-ACP. Chain termination also may occur when fatty acids are transferred from ACP to glycerol-3-phosphate by acyltransferases (Ohlrogge et al., 1993) (see below). 

ACP is rapidly hydrolyzed to free fatty acids. These authors have suggested a possible role for acyl-ACP thioesterase as one component of a switching system which allows indirectly acyl transfer from ACP to CoA derivatives in chloroplasts. The long-chain acyl-CoA synthetases which catalyze the following reaction (Fig. 10) ... 

Fig. 11. Various hypotheses proposed by which higher plants may attain high levels of unsaturated fatty acids in their chloroplast membrane galactolipids. (a) Phosphatidylcholine acts as a carrier molecule involved in the desaturation, (b) Desaturation of fatty acids occurs after formation of the galactolipid molecule, (c) Desaturation occurs before formation of the galactolipid molecule. In the first hypothesis, all the desaturases involved are confined in the chloroplast in the second hypothesis, the conversion of 18 1 to 18 2 is maximal in microsomes," whereas desaturation of 18 2 to 18 3 is highest in chloroplast membranes, (d) Deacylation-reacylation mechanism in which X can be a CoA-thioester, a polar lipid, etc. D, Desaturases T, acyl-ACP thioesterase e.r., endoplasmic reticulum.

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