Acetyl-ACP

The enzymes that catalyze formation of acetyl-ACP and malonyl-ACP and the subsequent reactions of fatty acid synthesis are organized quite differently in different organisms. We first discuss fatty acid biosynthesis in bacteria and plants, where the various reactions are catalyzed by separate, independent proteins. Then we discuss the animal version of fatty acid biosynthesis, which involves a single multienzyme complex called fatty acid synthase. 

The individual steps in the elongation of the fatty acid chain are quite similar in bacteria, fungi, plants, and animals. The ease of purification of the separate enzymes from bacteria and plants made it possible in the beginning to sort out each step in the pathway, and then by extension to see the pattern of biosynthesis in animals. The reactions are summarized in Figure 25.7. The elongation reactions begin with the formation of acetyl-ACP and malonyl-ACP, which... 

Steps 1-2 of Figure 29.5 Acyl Transfers The starting material for fatty-acid synthesis is the thioesteT acetyl CoA, the ultimate product of carbohydrate breakdown, as we ll see in Section 29.6. The synthetic pathway begins with several priming reactions, which transport acetyl CoA and convert it into more reactive species. The first priming reaction is a nucleophilic acyl substitution reaction that converts acetyl CoA into acetyl ACP (acyl carrier protein). The reaction is catalyzed by ACP transacyla.se. 

In bacteria, ACP is a small protein of 77 residues that transports an acyl group from enzyme to enzyme. In vertebrates, however, ACP appears to be a long arm on a multienzyme synthase complex, whose apparent function is to shepherd an acyl group from site to site within the complex. As in acetyl CoA, the acyl group in acetyl ACP is linked by a thioester bond to the sulfur atom of phosphopantetheine. The phosphopantetheine is in turn linked to ACP through the side-chain -OH group of a serine residue in the enzyme. 

The sex pheromone is interesting from a biosynthetic perspective (see Fig. 4.3) because it is closely connected with primary metabohsm. That is, the monomer 4 is an intermediate in fatty acid biosynthesis. Condensation of acetyl-ACP (8 ACP, acyl carrier protein) with malonyl-CoA (9 CoA, coenzyme A) yields acetoacyl-ACP (10). Enantioselective reduction with NADPH leads to (R)-3-hydroxybutyryl-ACP (11). Two units of this precursor could then be condensed to form the pheromone 5, which then degrades to 4 and 6 as described above. Alternatively, 4 can also be formed by direct hydrolysis of intermediate 11. 

Both bacteria and plants have separate enzymes that catalyze the individual steps in the biosynthetic sequence (Fig. 17-12). The fatty acyl group grows while attached to the small acyl carrier protein (ACP).54 58 Control of the process is provided, in part, by the existence of isoenzyme forms. For example, in E. coli there are three different P-oxoacyl-ACP synthases. They carry out the transfer of any acyl primer from ACP to the enzyme, decarboxylate malonyl-ACP, and carry out the Claisen condensation (steps b, e, and/in Eq. 17-12)58a e One of the isoenzymes is specialized for the initial elongation of acetyl-ACP and also provides feedback regulation.58c The other two function specifically in synthesis of unsaturated fatty acids. 

Outline of the reactions for fatty acid biosynthesis. Fatty acids grow in steps of two-carbon units and take place on a multienzyme complex, (a) The initial reactions of fatty acid biosynthesis are shown. In the first reaction, acetyl-CoA reacts with ACP (acyl carrier protein) to form acetyl-ACP (step 1). ACP is shown with its SH group emphasized (see fig. 18.13) to remind readers that the acyl derivatives are linked to ACP via a thioester bond. Malonyl-CoA, derived from the carboxylation of acetyl-CoA (see fig. 18.9), reacts... 

Citrate lyase catalyzes the cleavage of citrate to oxaloacetate and acetate in the presence of Mg2+ or Mn2+, but in the presence of EDTA it catalyzes its synthesis. The enzyme is a complex of three subunits. The y-subunit functions as an acyl carrier protein (ACP). The a-subunit is an acyl transferase involved in citryl-ACP formation and the release of acetate, and the /8-subunit catalyzes the cleavage of the citryl-ACP intermediate to oxaloacetate and acetyl-ACP. The enzyme from Klebsiella aerogenes has been purified, and binds 18 Mn2"1 in a cooperative manner. 

Figure 8 Decarboxylation of malonyl-ACP by the CLF. Decarboxylation of malonyl-ACP by the CLF results in acetyl-ACP, an enzyme-bound starter unit. Initiation of chain extension can then occur by transfer of the acetate starter unit to the KS domain.

The pathway The first committed step in fatty acid biosynthesis is the carboxylation of acetyl CoA to form malonyl CoA which is catalyzed by the biotin-containing enzyme acetyl CoA carboxylase. Acetyl CoA and malonyl CoA are then converted into their ACP derivatives. The elongation cycle in fatty acid synthesis involves four reactions condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP releasing free ACP and C02, then reduction by NADPH to form D-3-hydroxybutyryl-ACP, followed by dehydration to crotonyl-ACP, and finally reduction by NADPH to form butyryl-ACP. Further rounds of elongation add more two-carbon units from malonyl-ACP on to the growing hydrocarbon chain, until the C16 palmitate is formed. Further elongation of fatty acids takes place on the cytosolic surface of the smooth endoplasmic reticulum (SER). 

CoA to form malonyl CoA using C02 in the form of bicarbonate HC03 (Fig. 2). This reaction is catalyzed by the enzyme acetyl CoA carboxylase which has biotin as a prosthetic group, a common feature in C02-binding enzymes. One molecule of ATP is hydrolyzed in the reaction, which is irreversible. The elongation steps of fatty acid synthesis all involve intermediates linked to the terminal sulfhydryl group of the phosphopantetheine reactive unit in ACP phosphopantetheine is also the reactive unit in CoA. Therefore, the next steps are the formation of acetyl-ACP and malonyl-ACP by the enzymes acetyl transacylase and malonyl transacylase, respectively (Fig. 2). (For the synthesis of fatty acids with an odd number of carbon atoms the three-carbon propionyl-ACP is the starting point instead of malonyl-ACP.)... 

Condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP, releasing free ACP and C02 (catalyzed by acyl-malonyl-ACP condensing enzyme). 

To test this hypothesis, a number of KS-CLF mutants have been prepared and their ability to decarboxylate malonyl ACP determined. The results are summarised in Figure 10. In Figure 10a, we have the ESMS transform of the initial malonyl ACP. When this is incubated with KS(ala)/CLF(glu) in which the KS cysteine has been mutated so that no polyketide production is possible, we see slow conversion of malonyl ACP to acetyl ACP as predicted. The time course for the decarboxylation is shown below and this indicates ca. 40% decarboxylation after 1 hour. Intriguingly, when the malonyl ACP is incubated with a KS(glu)/CLF(ala) mutant, we see very rapid and complete decarboxylation. A similar result has been reported by Smith and co-workers with the mammalian FAS KS. ... 

Figure 10 Conversion of malonyl-ACP to acetyl-ACP and polyketide production by KS/CLF mutants, (a) ESMS analysis of initial sample of malonyl-ACP before decarboxylation and after incubation with either (middle) KS(ala)/CLF(gln) (60 min), or (right) KS(gln)/CLF(ala) (1 min), (b) Time course of decarboxylation of malonyl CoA by KS(ala)/CLF(gln), and production of octaketides (SEK-4 + SEK-4b) from malonyl ACP by KS(cys)/CLF(ala), both in the presence (triangles) and absence (squares) of acetyl-ACP.

When treated with a KS(aIa)/CLF(ala) mutant, no decarboxylation occurred and the malonyl ACP was recovered unchanged. Thus these experiments appear to indicate that the role of the CLF is, at least in part, to provide the acetyl ACP needed to initiate chain assembly. The significance of this result is that if malonyl CoA is the immediate source of both the chain-initiating unit and the chain starter, experiments with wild-type proteins to produce novel metabolites with alternate starter units would not be feasible. However, by impairing the ability of the minimal PKS to produce its own starter, the uptake of added alternate starter units will be facilitated. This may still be a problem because it has been shown that the KS retains a decarboxylase activity and a KS(cys)/CLF(ala) mutant is still capable of producing polyketides, albeit at a greatly reduced level. However, as shown in Figure 10b, addition of acetyl ACP to this restores the rate of polyketide production. 

The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP and malonyl ACP. Acetyl transacylase and malonyl transacylase catalyze these reactions. 

In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to form a C5-P-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C4-P-ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C5-P-ketoacyl ACP into a C5-acyl ACP, which is ready for a third round of elongation. The elongation cycles continue until Ci5-acyl ACP is formed. This intermediate is a good substrate for a thioesterase that hydrolyzes C 15-acyl ACP to yield palmitate and ACP. The thioesterase acts as a ruler to determine fatty acid chain length. The synthesis of longer-chain fatty acids is discussed in Section 22.6. 

Decarboxylation drives the condensation of malonyl ACP and acetyl ACP. In contrast, the condensation of two molecules of acetyl ACP is energetically unfavorable. In gluconeogenesis, decarboxylation drives the formation of phosphoenolpyruvate from oxaloacetate. 

CH3COSC0A -b HCOJ -P ATP HO2CCH2COSC0A -b Pf -b ADP Conversion of acetyl-ACP to butanoyl-ACP (four-step cycle)... 

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