CoA thioester, formation

Oxidoreductases CoA thioester formation through aldehyde oxidation 384... 

Enzymology of the Formation of Hydroxyacyl-CoA Thioesters as Substrates for PHA Synthases... 

Alkaloids 36-41 were isolated from Lupinus luteus L. seedlings. They are considered to be lupinine esters with 4-hydroxycinnamic acids (94-100). The structures of these new alkaloids were elucidated on the basis of H NMR, MS, and chemical and enzymatic transformations. All these alkaloids were obtained from lupinine and hydroxycinnamic acid by two enzymatic systems (96-97) ligase catalyzed formation of the CoA-thioester, and transferase catalyzed lupinine ester formation from the CoA-thioester. 

Like the related fatty acid synthases (FASs), polyketide synthases (PKSs) are multifunctional enzymes that catalyze the decarboxylative (Claisen) condensation of simple carboxylic acids, activated as their coenzyme A (CoA) thioesters. While FASs typically use acetyl-CoA as the starter unit and malonyl-CoA as the extender unit, PKSs often employ acetyl- or propionyl-CoA to initiate biosynthesis, and malonyl-, methylmalonyl-, and occasionally ethylmalonyl-CoA or pro-pylmalonyl-CoA as a source of chain-extension units. After each condensation, FASs catalyze the full reduction of the P-ketothioester to a methylene by way of ketoreduction, dehydration, and enoyl reduction (Fig. 3). In contrast, PKSs shortcut the FAS pathway in one of two ways (Fig. 4). The aromatic PKSs (Fig. 4a) leave the P-keto groups substantially intact to produce aromatic products, while the modular PKSs (Fig. 4b) catalyze a variable extent of reduction to yield the so-called complex polyketides. In the latter case, reduction may not occur, or there may be formation of a P-hydroxy, double-bond, or fully saturated methylene additionally, the outcome may vary between different cycles of chain extension (Fig. 4b). This inherent variability in keto reduction, the greater variety of... 

The biosynthesis of polyketides is analogous to the formation of long-chain fatty acids catalyzed by the enzyme fatty acid synthase (FAS). These FASs are multi-enzyme complexes that contain numerous enzyme activities. The complexes condense coenzyme A (CoA) thioesters (usually acetyl, propionyl, or malonyl) followed by a ketoreduction, dehydration, and enoylreduction of the [3-keto moiety of the elongated carbon chain to form specific fatty acid products. These subsequent enzyme activities may or may not be present in the biosynthesis of polyketides. 

Scheme 36 represents the first example for a [NiS] mediated formation of thioesters from alkyl, CO, and thiol groups in a cyclic way. All intermediates shown in Scheme 37 could be intercepted and characterized by spectroscopic methods and X-ray structure analysis. They permit a detailed insight into the individual steps of thioester formation that may take place in an analogous way at the active site of CODH when acetyl-CoA is synthesized. 

The peanut chalcone synthase and parsley stilbene synthases have been cloned, expressed in E. colt, and purified to homogeneity [135,137]. The enzymes appear to be mechanistically similar each catalyzes the formation of a tetraketide from three molecules of malonyl CoA that are decarboxylated and condensed with a starter unit derived from p-coumaroyl CoA or a similar CoA thioester (Fig. 6). No reductions or dehydrations occur during either chalcone or stilbene synthesis, and some products spontaneously cyclize following their release from the enzyme. A major feature that distinguishes chalcone and stilbene synthases is that the latter perform an additional decarboxylation to remove a carbon atom that is present in chalcone products [132,138]. The presence of this additional carboxyl group results in a different cyclization pattern for chalcone products. The precise mechanisms by which chalcone and stilbene synthases determine the fate of this carbon atom are not known. 

It is usual in humans for the S(+)-enantiomer of 2-arylpropionic acids to predominate in plasma and for the S(- -)- to R(-)-enantiomeric ratio of plasma concentrations to increase with time after administration of the racemate, which is often attributed to metabolic inversion of the chiral center of the R( )-enantiomers to their S(- -)-antipodes. ° In humans, the S(- -)-enantiomer is generally eliminated more slowly than is the R( )-enantiomer. The extent of chiral inversion of fenoprofen, which has been attributed to the differential rate of formation of the CoA-thioester by hepatic microsomes, varies widely among species. It has been estimated to be 90% in dogs, 80% in sheep, 73% in rabbits, 60% in humans, 42% in rats,f and 38% in horses. ... 

FIGURE 32.10 A few examples of reactions of acetylation (a), and of some reactions consecutive to the formation of xenobiotic acyl-CoA conjugates (b). The substrates are salicylic acid (19), (R)-ibuprofen (21), and valproic acid whose intermediate acyl-CoA thioester is shown here (22). The arrows point to the target sites. 

A radiometric and a spectrometric assay have been developed to measure PhaC activity. The radiometric assay measures the incorporation of isotope-labeled hydroxyacyl moieties into the polyester, which is present from the beginning as primer [17]. [3-14]ft-(-)-3-hydroxybutyryl-CoA or [3H]-P,S-3-hydroxybutyryl-CoA or in principle any other CoA thioester of a radioactively-labeled hydroxyacyl moiety could be used as substrate. Only the radioactivity that is really incorporated into the insoluble polyester is measured. The time course of the assay, the need to synthesize the substrates and the high costs make the assay very inconvenient and it is hardly used anymore. A more convenient assay is the spectrometric assay which measures the release of coenzyme A during the polymerization reaction in presence of Ellmann s reagent 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) yielding 5 -thio(2-nitrobenzoate) that absorbs at about 412 nm [16], Here, the enzyme activity is measured directly without delay. However, it is not the formation of the polymeric product that is measured, but the release of coenzyme A, which can also be due to the hydrolytic cleavage of the substrate by another enzyme that does not have any PhaC activity, like a thioesterase. Nevertheless, this assay is now most frequently used due to its convenience. 

Neither El nor E2, together or alone, mediated the formation of acrylyl- or lactyl-CoA from the respective acid plus CoA and acetyl phosphate or acetyl-CoA. However, a CoA transferase that catalyzes the formation of the CoA thioesters of lactate, acrylate, and propionate from acetyl-CoA has been isolated from C. propionicum (233). It is curious that acrylyl-CoA could not be isolated as a product with the crude or purified enzyme system, although acrylyl-CoA is readily converted to lactyl-CoA in the purified system. Abeles suggested that perhaps acrylyl-CoA exists as an enzyme-bound intermediate that requires the presence of a reductase for release as propionate. In this manner the organism is protected from buildup of toxic amounts of any acrylyl intermediates, which are known to undergo Michael addition reactions with biological nucleophiles. 

The acyl-CoA ligases (EC 6.2.1.- often also referred to as acyl-CoA synthetases, or ACSs) catalyze the reversible nucleoside triphosphate-dependent formation of acyl-CoA thioesters from CoA and a free carboxylic acid. Two mechanistic types can be distinguished in this group of enzymes the first uses ATP to activate... 

Scheme 8 Two mechanistic proposals for the catalytic mechanism of CoA-transferases. In mechanism A, an acyl-enzyme Intermediate Is formed by reaction of an enzyme-bound glutamate (aspartate for Class III enzymes) with the donor acyl-CoA, followed by the formation of an enzyme-bound glutamyl- (or aspartyl-) CoA thioester Intermediate. The thioester subsequently reacts with the acceptor carboxylate to give a new acyl-enzyme anhydride from which the acyl group Is transferred to CoA. In Class I transferases, this process follows classical ping-pong kinetics, whereas In Class III enzymes the donor carboxylate only leaves the enzyme complex upon formation of the product (see text for details). Mechanism B represents a ternary complex mechanism as used by Class II enzymes In which a transient anhydride made up of the donor and acceptor acyl groups Is formed by reaction of the acceptor carboxylate with the donor acyl-ACP. The free ACP subsequently reacts with this anhydride to complete acyl transfer.

The metabolism of fatty acids requires their prior activation by conversion to fatty acyl-CoA thioesters. The activating enzymes are ATP-dependent acyl-CoA synthetases, which catalyze the formation of acyl-CoA by the following two-step mechanism in which E represents the enzyme ... 

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