Coenzyme A thioester

Several Clostridia including Cl. tyrobutyricum. Cl. thermoaceticum, and Cl. kluyveri are able to reduce the double bond of a,p-unsaturated aldehydes, ketones, and coenzyme A thioesters (Rohdish et al. 2001), and are noted in Chapter 3, Part 3. 

Formation of the coenzyme A thioester is mediated by a ligase, which has been demonstrated in a number of organisms including Pseudomonas sp. strain K172... 

Pathways Providing Coenzyme A Thioesters of other HASCL.104... 

PHA synthases are the key enzymes of PHA biosynthesis. These enzymes catalyze the covalent linkage between the hydroxyl group of one and the carboxyl group of another hydroxyalkanoic acid. The substrates of PHA synthases are the coenzyme A thioesters of hydroxyalkanoic acids there is no evidence that PHA synthases can utilize either free hydroxyalkanoic acids or other derivatives of hydroxyalkanoic acids. With respect to size, structure, and substrate specificity, three different types of PHA synthases (I, II, and III) can be distinguished (see below). 

PHA synthases exhibit a remarkably low substrate specificity. In general, the specificity of PHA synthases regarding the length of the hydroxyalkyl moiety of the coenzyme A thioester as well as of the position of the hydroxyl group, the presence of other substituents, and the position of double bonds, is low. In contrast, the stereospecificity of PHA synthases is strict the hydroxyl carbon atom must have the R-configuration. 

The presence of a PHA synthase alone is not sufficient to allow synthesis of PHAs. PHA biosynthesis will not occur if genes encoding enzymes required for the synthesis of hydroxyacyl-coenzyme A thioesters are absent or if the pathways constituted by these enzymes are for whatever reason not functionally active. This becomes evident, for example, when a PHA synthase gene is expressed in wild type or normal laboratory strains of E. coir, even if a functionally active PHA synthase is expressed, no or only traces of PHAs are accumulated. 

There are an overwhelming number of studies which successfully demonstrated heterologous expression of PHA synthesis genes in bacteria it will therefore not be possible to mention them all. Establishment of functional active PHA biosynthesis pathways in E. coli requires not only a PHA synthase but also enzymes that allow the conversion of metabolites, which derive from the provided carbon source, into the R isomers of hydroxyacyl-coenzyme A thioesters that are used as a substrate by the respective PHA synthase. Otherwise, no or only marginal amounts of PHAs are accumulated. 

In addition to the aspects mentioned above, the extent of PHA accumulation might depend on quite different features. The only physiological inhibitor of PHA synthase that has been identified is coenzyme A as pointed out in an earlier section. Since the concentration of coenzyme A in the cytoplasma will probably never rise to very high levels, it may be questionable whether this inhibition is physiologically relevant at all. In our opinion, the availability of a hy-droxyacyl coenzyme A thioester provided by the biosynthesis pathway is most important to initiate PHA biosynthesis (compare also [8] - this book). 

Thioesters play a paramount biochemical role in the metabolism of fatty acids and lipids. Indeed, fatty acyl-coenzyme A thioesters are pivotal in fatty acid anabolism and catabolism, in protein acylation, and in the synthesis of triacylglycerols, phospholipids and cholesterol esters [145], It is in these reactions that the peculiar reactivity of thioesters is of such significance. Many hydrolases, and mainly mitochondrial thiolester hydrolases (EC 3.1.2), are able to cleave thioesters. In addition, cholinesterases and carboxylesterases show some activity, but this is not a constant property of these enzymes since, for example, carboxylesterases from human monocytes were found to be inactive toward some endogenous thioesters [35] [146], In contrast, allococaine benzoyl thioester was found to be a good substrate of pig liver esterase, human and mouse butyrylcholinesterase, and mouse acetylcholinesterase [147],... 

K. M. Knights, R. Gasser, W. Klemisch, In vitro Metabolism of Acitretin by Human Liver Microsomes Evidence for an Acitretinoyl-Coenzyme A Thioester Conjugate in the Transesterification to Etretinate , Biochem. Pharmacol. 2000, 60, 507-516. 

Figure 4. Chemical synthesis of galloyl coenzyme A thioester (4). (1) 4-0-/3-D-glucosidogallic acid (2) AT-succinimidyl 4-0-/3 -D-glucosidogallate (3)...

The mechanism for bacterial synthesis of PHA is not the simple dehydration reaction between alcohol and carboxyl groups. It is more complicated and involves the coenzyme A thioester derivative of the hydroxyalkanoic acid monomer (produced from the organic feedstock available to the bacteria) [Kamachi et al., 2001], Growth involves an acyl transfer reaction catalyzed by the enzyme PHA synthase (also called a polymerase) [Blei and Odian,... 

Lipases are enzymes that catalyze the in vivo hydrolysis of lipids such as triacylglycerols. Lipases are not used in biological systems for ester synthesis, presumably because the large amounts of water present preclude ester formation due to the law of mass action which favors hydrolysis. A different pathway (using the coenzyme A thioester of a carboxylic acid and the enzyme synthase [Blei and Odian, 2000]) is present in biological systems for ester formation. However, lipases do catalyze the in vitro esterification reaction and have been used to synthesize polyesters. The reaction between alcohols and carboxylic acids occurs in organic solvents where the absence of water favors esterification. However, water is a by-product and must be removed efficiently to maximize conversions and molecular weights. 

The fourth and last step of the /3-oxidation cycle is catalyzed by acyl-CoA acetyltransferase, more commonly called thiolase, which promotes reaction of /3-ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA The other product is the coenzyme A thioester of the fatty acid, now shortened by two carbon atoms (Fig. 17-8a). This reaction is called thiolysis, by analogy with the process of hydrolysis, because the /3-ketoacyl-CoA is cleaved by reaction with the thiol group of coenzyme A... 

Following removal of one acetyl-CoA unit from palmitoyl-CoA, the coenzyme A thioester of the shortened fatty acid (now the 14-carbon myristate) remains. The myristoyl-CoA can now go through another set of four /3-oxidation reactions, exactly analogous to the first, to yield a second molecule of acetyl-CoA and lauroyl-CoA, the coenzyme A thioester of the 12-carbon laurate. Altogether, seven passes through the j8-oxidation sequence are required to oxidize one molecule of palmitoyl-CoA to eight molecules of acetyl-CoA (Fig. 17-8b). The overall equation is... 

Fatty acid biosynthesis. Activated as its coenzyme A thioester, the growing fatty acid (acyl-CoA) acylates malonyl-CoA in a malonic ester synthesis. Two carbon atoms are added, with the third lost as C02. Enzymatic reduction, dehydration, and further reduction gives a fatty acid that has been lengthened by two carbon atoms. 

Carboxylic acids. Aliphatic carboxylic acids (R—GOOH) are deprotonated at physiological pH (pH 7) and are therefore represented as R—COO. Thus, acetic acid (GHj—COOH) exists as acetate (CH3COO ) at pH 7. A variety of short chain mono-, di-and tricarboxylic acids are important intermediates in metabolism and may be present at low concentrations in all cells either as the acid or as a covalent adduct. Thus, acetate (C2) and malonate (C3) can exist as the key acyl-coenzyme A thioester intermediates acetylGoA and malonylCoA, respectively. Phosphoenolpyruvate (C3), 1,3-bisphosphoglyceric acid (C3) and 3-phosphoglyc,erate (C3) are key metabolic intermediates. 

According to the above-mentioned hypothesis, the caffeic acid moiety is retransferred to coenzyme A for further modification reactions. Methylation of the caffeoyl moiety in position 3 is achieved by S-adenosyl-L-methionine (SAM)-dependent 0-methyltransferases (OMTs) either acting on the level of the free acid or the coenzyme A thioester. Hydroxylation in position 5 is catalysed by a cytochrome P450 of the CYP84 family which will be described in more detail. Establishment of the sinapoyl substitution pattern by adding another methyl group will be depicted below. 

Strack, D., Gross, W., Heilemann, J., Keller, H. and Ohm, S. (1988) Enzymic synthesis of hydroxycinnamic acid esters of glucaric acid and hydroaromatic acids from the respective 1-O-hydroxycinnamoylglucoside and hydroxycinnamoyl-coenzyme-A thioester as acyldonors with a protein preparation from Cestrum elegans leaves. Z. Naturforsch., 43c, 32-6. 

Polyketide and fatty acid biosyntheses begin with condensation of the coenzyme A thioester of a short-chain carboxylic acid starter unit such as acetate or propionate with the coenzyme A thioester of a dicarboxylic acid extender unit such as malonate or methyl malonate. The driving force for the condensation is provided by the decarboxylation of the extender unit. In the case of fetty acid synthesis, the resulting -carbonyl is completely reduced to a methylene however, during the synthesis of complex poly-ketides, the -carbonyl may be left untouched or variably reduced to alcohol, olefinic, or methylene functionalities depending on the position that the extender unit will occupy in the final product. This cycle is repeated, and the number of elongation cycles is a characteristic of the enzyme catalyst. In polyketide biosynthesis, the full-length polyketide chain cyclizes in a specific manner, and is tailored by the action of additional enzymes in the pathway. 

Holden HM, Banning MM, Haller T, Gerlt JA. The crotonase superfamily divergently related enzymes that catalyze different reactions involving acyl coenzyme A thioesters. Acc. Chem. Res. 2001 34 145-157. 

An unique example of stereoisomer selective tissue uptake of stereoisomers is noted with NSAIDs. The R enantiomer of ibuprofen shows preferential uptake into fat following the administration of the racemate and individual isomersJ However, this apparent difference in fat distribution is probably a consequence of the selective metabolic uptake and formation of the coenzyme A thioester of the R isomer, which does not occur with the S isomer. 

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