Acyl-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. 

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... 

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. 

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. 

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]. 

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.

Fatty acid synthase (FAS) constitutes a multisubunit complex in the plastid where it catalyzes ordered synthesis of fatty acids, initiated from acetyl CoA and malonyl CoA [146, 147]. Stepwise FAS activity generates the products 16 0-acyl carrier protein (AGP) and 18 0-ACP. Most of the 18 0-ACP is desaturated by a soluble stearoyl-ACP desaturase, yielding 18 1 D9-ACP [148]. Acyl-ACP thioesterases release AGP from 16 0-AGP and 18 0-AGP the deacylated fatty acids exit the plastid and are then esterified with coenzyme A (GoA) to form respective acyl-GoAs [148]. These acyl moieties are then esterified to phosphatidylcholine (PQ and then undergo desaturation by D12- and D15-desaturases to yield the essential fatty acids, linoleic acid, and a-linolenic acid [148-150], All higher plants have the enzymes for synthesizing the G18 PUFAs linoleic acid and a-linolenic acid. The primary genes involved in PUFA biosynthesis have been reviewed [151],... 

BROAD-RANGE AND BINARY-RANGE ACYL-ACP THIOESTERASES FROM MEDIUM-CHAIN PRODUCING SEEDS... 

Broad-Range and Binary-Range Acyl-ACP Thioesterases from Medium-Chain Producing Seeds. 

Acyl-ACP thioesterase. The factors involved in the termination of fatty acid biosynthesis in the plastid at the level of palmitoyl-ACP, stearoyl-ACP and oleoyl-ACP are largely unknown, although several possibilities have been considered (Harwood, 1988 Stumpf, 1989 Slabas and Fawcett, 1992). Primary importance, however, has been attached to selective hydrolysis of the acyl-ACPs involving specific acyl-ACP thioesterases (EC 3.1.2.14) (Hills and Murphy, 1991). Systems with particular preference for the hydrolysis of palmitoyl-ACP and oleoyl-ACP... 

Pollard, M.R., Anderson, L., Fan, C., Hawkins, D.J. and Davies, H.M. (1991) A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia califomica. Arch, Biochem. Biophys. 284, 306-312. 

Thioesterase activity has also been targeted as a means of producing medium-chain saturated fatty acids (MCFA) in BOS. MCFA, such as lauric acid (12 0), are widely used for industrial applications, including cosmetics and surfactants, as well as for certain food applications such as confectionary. In most BOS, including canola, the endogenous acyl-ACP thioesterase exhibits a preference for 18-carbon FAs. Production of MCFA required the introduction of a thioesterase with appropriate medium-chain specificity, such as the 12 0 ACP-thioesterase from the California bay laurel (Umbellularia californica) (Voelker et al., 1996). Although the expression of this enzyme in canola resulted in a relatively high accumulation of lauric acid. 

Hawkins, D.J. and Kridl, J.C. 1998. Characterization of acyl-ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola. Plant J. 13 743-752. 

T. A. McKeon and P. K. Stumpf, Purification and characteri2 ation of the stearoyi-ACP desaturcise emd the acyl-ACP thioesterase from maturing seeds of safflower, 3. Biol. Chem. 257 12141 (1982). 

---