CoA thioesters

FIGURE 24.8 The mechanism of the acyl-CoA synthetase reaction involves fatty acid carboxylate attack on ATP to form an acyl-adenylate intermediate. The fatty acyl CoA thioester product is formed by CoA attack on this intermediate. 

Reaction 3 is analogous to the dehydrogenation of fatty acyl-CoA thioesters (see Figure 22—3). In isovaleric acidemia, ingestion of protein-rich foods elevates isovalerate, the deacylation product of isovaleryl-CoA. Figures 30-20, 30-21, and 30-22 illustrate the subsequent reactions unique to each amino acid skeleton. 

Particularly important to the pathways of modular synthases is the incorporation of novel precursors, including nonproteinogenic amino acids in NRP systems [17] and unique CoA thioesters in PK and fatty acid synthases [18]. These building blocks expand the primary metabolism and offer practically unlimited variability applied to natural products. Noteworthy within this context is the contiguous placement of biosynthetic genes for novel precursors within the biosynthetic gene cluster in prokaryotes. Such placement has allowed relatively facile elucidation of biosynthetic pathways and rapid discovery of novel enzyme mechanisms to create such unique building blocks. These new pathways offer a continued expansion of the enzymatic toolbox available for chemical catalysis. 

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

Defects of mitochondrial transport interfere with the movement of molecules across the inner mitochondrial membrane, which is tightly regulated by specific translocation systems. The carnitine cycle is shown in Figure 42-2 and is responsible for the translocation of acyl-CoA thioesters from the cytosol into the mitochondrial matrix. The carnitine cycle involves four elements the plasma membrane carnitine transporter system, CPT I, the carnitine-acyl carnitine translocase system in the inner mitochondrial membrane and CPT II. Genetic defects have been described for each of these four steps, as discussed previously [4,8,9]. 

Structures of CHS complexed with different Coenzyme A (CoA) thioesters and product analogs (i.e., naringenin and resveratrol) demonstrate that the active site is buried within an interior cavity located in the cleft between the upper and lower domains of each monomer (Fig. 12.3). Considering the complexity of the reaction... 

Each CHS monomer consists of two structural domains (Fig. 12.5, left). The upper domain exhibits the a-p-a-p-a pseudo-symmetric motif observed in fatty acid P-ketoacyl synthases (KASs) (Fig. 12.5, right).20 Both CHS and KAS use a cysteine as a nucleophile in the condensation reaction, and shuttle reaction intermediates via CoA thioester-linked molecules or ACPs, respectively. The conserved architecture of the upper domain maintains the three-dimensional position of the catalytic residues of each enzyme Cysl64, His303, and Asn336 in CHS correspond to a Cys, His, and His in KAS I and II. 

Figure 12.6 Starter molecule engineering. A. Reaction catalyzed by ACS. B. Thin layer chromatography screening for enzymatic activity with different starter molecules. C. Views illustrate the active site of the F215S mutant (right), wild-type CHS with N-methylanthraniloyl-CoA (center), and wild-type CHS with p-coumaroyl-CoA (left) modeled at the active site entrances. The catalytic residues, Cys 164, His 303, and Asn 336, and Phe 265 are shown. In wild-type CHS, N-methylanthraniloyl-CoA clashes with Phe 215 to prevent the CoA thioester from adopting the conformation depicted in (A). The wild-type - p-coumaroyl-CoA model emphasizes the ability of the propanoid linker to extend the phenolic ring deeper into the active site cavity.

Our results demonstrate that type III PKSs use multiple regiochemical mechanisms for achieving specificity during the loading of a given CoA-thioester... 

The first STS structure solved was that of a pinosylvin-forming STS from Pinus sylvestris. Pine trees can by-pass the C4H reaction and directly produce the CoA thioester of cinnamate that allows this STS to utilize a non-substituted cinnamoyl-CoA starter in vz vo.11 However, when presented in vitro with p-coumaroyl-CoA, the enzyme proves to be comparable in activity to STS enzymes from organisms that utilize the para-substituted cinnamoyl starter. 

Boelsterli UA. Xenobiotic acyl glucuronides and acyl CoA thioesters as protein-reactive metabolites with the potential to cause idiosyncratic drug reactions. Curr Drug Metab 2002 3(4) 439-450. 

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. 

Once inside cells, fatty acids are activated at the outer mitochondrial membrane by conversion to fatty acyl-CoA thioesters. Fatty acyl-CoA to be oxidized enters mitochondria in three steps, via the carnitine shuttle. 

Polycarboxylic acid synthases. Several enzymes, including citrate synthase, the key enzyme which catalyzes the first step of the citric acid cycle, promote condensations of acetyl-CoA with ketones (Eq. 13-38). An a-oxo acid is most often the second substrate, and a thioester intermediate (Eq. 13-38) undergoes hydrolysis to release coenzyme A.199 Because the substrate acetyl-CoA is a thioester, the reaction is often described as a Claisen condensation. The same enzyme that catalyzes the condensation of acetyl-CoA with a ketone also catalyzes the second step, the hydrolysis of the CoA thioester. These polycarboxylic acid synthases are important in biosynthesis. They carry out the initial steps in a general chain elongation process (Fig. 17-18). While one function of the thioester group in acetyl-CoA is to activate the methyl hydrogens toward the aldol condensation, the subsequent hydrolysis of the thioester linkage provides for overall irreversibility and "drives" the synthetic reaction. 

Figure 1 Polyketide biosynthesis. Polyketide backbones are formed via condensations from acyl-CoA thioesters of carboxylic acids. The (3-ketone which results from each condensation can undergo a series of reductive steps analogous to fatty acid biosynthesis. However, either none or only some of the reductive activities may occur in a given cycle. This allows PKSs to generate diversity through selection of priming and extender units, variation of the reductive cycle, and stereoselectivity. (ACP, acyl carrier protein AT, acyl transferase KS, ketosynthase DH, dehydratase ER, enoylreductase KR, ketoreductase TE, thioesterase.) The structure depicted in the lower right-hand corner is representative of the possible structural variations that can arise during polyketide biosynthesis.

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... 

Based on the identified homology of the cefD and cefDZ proteins with known eukaryotic enzymes, a mechanism for the A. chrysogenum two-component epimerization system which is different from the epimerization found in prokaryotes has been established <2002JBC46216>. Therefore, it was suggested that the cephalosporin biosynthesis pathway begins with the activation of the substrate isopenicillin N to its CoA, followed by an epimerization to the D-enantiomer, namely penicillinyl-CoA. Next, the required hydrolysis of the CoA-thioesters seems to occur in a nonstereoselective manner by different thioesterases. The resulting product, penicillin N, is the direct precursor of all cephalosporins and cephamycins. 

Whereas we have no intention to describe in this review the various aspects of halobacterial metabolism, we would like to mention several unique features of their metabolic system. The conversions of the two 2-oxoacids (pyruvate and oxoglutarate) to their corresponding acyl-CoA thioesters are crucial steps in the two pathways described above. In most eukaryotes and aerobic eubacteria these reactions are catalyzed by the 2-oxoacid dehydrogenase multienzyme complexes that use NAD+ as the final electron acceptor. These complexes are... 

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. 

Methylmalonyl-CoA epimerase shifts the CoA thioester from C-l (of the original propionyl group) to the newly added carboxylate, making the product L-methylmalonyl-CoA. 

Yu and coworkers measured the substrate specificity of rat liver mitochondrial thioesterase, which hydrolyzes acyl-CoA to CoA and free fatty acid (see Chapter 21). This enzyme was approximately twice as active with C14-CoA thioesters as with Ci8-CoA thioesters. 

Fatty acid oxidation is a multistep process requiring orchestration of reactions in the cytoplasm and mitochondria (Fig. 9-1). Free fatty acids enter the cell and are activated to their coenzyme A (CoA) thioesters in the reaction catalyzed by fatty acyl-CoA synthetase ... 

Now transported to the liver, fatty acids activate Giving CoA thioesters, oxidation is their fate Ketone bodies, Ketone bodies, because low glycerol-P Glucagon up, insulin down, stops reversal to TG. 

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