Cytosolic thioesterase

Once the 16-carbon atom acyl chain is formed, the thioester link between the acyl group and the 4 -phosphopante-theine of the carrier protein is hydrolysed by a thioesterase and palmitate is released. Synthesis of shorter-chain fatty acids, e.g. myristic (a C-14 carbon acid), requires a specific cytosolic thioesterase (thioesterase II) which is present in liver. It hydrolyses the thioester bond when fatty acids reach lengths of less than 16 carbon atoms. 

Both goat and cow mammary tissue synthesize medium-chain fatty acids. However, attempts to isolate thioesterase II from the cytosol of ruminant mammary tissues have not been successful (Grunnet and Knudsen 1979). In contrast to the nonruminant, the fatty acid-... 

Purified MCBP was incubated with [l-14C]myristoyl-CoA in separate tubes and aliquots were removed at selected times. At time 10 min, cytosolic fraction was added to one tube and buffer was added to the other. Aliquots were removed at selected times up to 120 min. The vial which did not contain added cytosol exhibited the formation of a protein-myristoyl-CoA complex, while the tube which contained added cytosol exhibited deacylation (Figure 17.4B). These results suggest that the cytosolic fraction may contain thioesterases/proteinases which could modulate the acylation reaction in vivo (Raju and Sharma 1997). The absence of acyl-complex formation in the cytosol could be due to the presence of either esterases or pro-teinases. It has been reported that porcine phospholipase A contained thioesterase and deacylase activities (Nocito et al. 1996). 

The answer is d. (Murray, pp 230-267. Scriver, pp 2297-2326. Sack, pp 121-138. Wihon, pp 287-320.) In humans, the end product of fatty acid synthesis in the cytosol is palmitic acid. The specilicity of cytosolic multienzyme, single-protein fatty acid synthetase is such that once the C16 chain length is reached, a thioesterase clips off the fatty acid. Elongation as well as desaturation of de novo palmitate and fatty acids obtained from the diet occur by the action of enzymes in the membranes of the endoplasmic reticulum. 

Two thioacyl protein thioesterases (APTs) have been identified. Unlike PATs, both are soluble proteins. Acylprotein thioesterase-1 (APTl) was purified from rat liver cytosol using palmitoylated G-protein a-subunit as a substrate (J.A. Duncan, 1998, 2002). This thioesterase, a 29-kDa monomeric protein, is likely to be the one involved in turnover of cytoplasmically disposed thioacyl groups of proteins. It displays both acylprotein thioesterase activity as well as lysophospholipase activity, but thioacylproteins are by far the preferred substrates. The second acylprotein thioesterase, protein palmitoylthioesterase-1 (L.A. Camp,... 

I also appears to possess lysophospholipase LI activity. Thioesterase II is a cytosolic tetrameric protein composed of 32-kDa subunits encoded by the tesB gene. Thioesterase... 

Lindquist, P.J.G., Svensson, L.T. Alexson, S.E.H. (1998) Enn J. Biochem. 251, 631-640. Molecular cloning of the peroxisome proliferator-induced 46-kDa cytosolic acyl-CoA thioesterase from mouse and rat liver. 

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]. 

Further desaturation and acyl chain elongation of fatty acids are generally accepted to involve cytosolic membranous systems associated with the endoplasmic reticulum (Stumpf, 1989). A critical importance, therefore, would appear to exist for a detailed understanding of the mechanics by which the fatty acids are exported from the plastid. At present, there is little available information on the precise transport system or its regulation. Although fatty acids are presumed to be released on hydrolysis of acyl-ACPs by specific thioesterases, it is not known whether an initial formation of acyl-CoA is required prior to transport across the plastidic membrane in association with carnitine or some other system. A direct transacylation between ACP and CoA or some carrier system would be less expensive energetically than a process involving hydrolysis and synthesis. 

It is now well established that the acyl-CoA thioesterase activity in rat and mouse liver is strongly induced by peroxisome proliferators. The induced activity is due to induction of several enzymes belonging to two gene families, type I and type II thioesterases, each of which contains several members of structurally related enzymes. The type I thioesterases are localized to cytosol, mitochondria and peroxisomes," " which... 

To redirect hydroxyvalerate production from PHA synthesis, they investigated the use of a Pseudomonas strain with systems for a removal of the CoA carriers from the hydroxy acids. Wild-type Pseudomonas overexpressing the gene of a recombinant thioesterase from E. coli yielded 14 g L 4-hydroxyvalerate and 5 g L 3-hydroxyvalerate as by-products (Fig. 12). They used minimal medium which was supplemented with glucose and levulinic acid (Martin and Prather 2009). Regarding the production of y-VL, they faced the problem that the intramolecular cyclization of 4-hydroxyvaleric acid is pH dependent and that the equilibrium at pH 7.5 in the cytosol is unfavorable for lactonization. By using a variant of human paraoxonase I, they achieved a substantial increase in y-VL production. 

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