P-Ketoacyl-ACP

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. 

Acetoacetyl ACP + NADPH + H+ d-3-hydroxybutyryl ACP + NADP P-Ketoacyl ACP reductase... 

This four-step cycle includes condensation of acetate and malonate to give ketobu-tanoate with subsequent reduction to butanoate in three further steps. These are reduction to the 3R hydroxy acid, dehydration to the 2t acid, and reduction again. Reduction is affected by NADPH and a proton. The process is then repeated to add further two-carbon units until a thioesterase liberates the free acid. This sequence requires a fatty acid synthase, which contains the enzymes needed for each of the four steps viz. p-ketoacyl-ACP synthase, p-ketoacyl-ACP reductase, p-ketoacyl-ACP dehydrase, and enoyl-ACP reductase, respectively. 

Figure 4 Modular organization of the six modules (I-VI) of 6-deoxyerythronolide B synthase (DEBS) enzyme as derived from Saccharopolyspora erythraea. Enzyme aetivi-ties are aeyltransferases (AT), aeyl carrier proteins (ACP), P-ketoacyl-ACP synthases (KS), P-ketoreductases (KR), dehytratases (DH), enoyl reductases (ER), and thioesterases (TE). The TE-catalyzed release of the polyketide chain results in the formation of 6-dEB (50) [212,216,217].

The last two carbons of the fatty acid chain (i.e., those most distal from the carboxylate group) are the first introduced into the nascent chain, and acetyl-CoA can be thought of as the primer molecule of fatty acid synthesis in E. coli. The initial condensation reaction, catalyzed by P-ketoacyl-ACP synthase III (FabH), utilizes acetyl-CoA and malonyl-ACP to form the four-carbon acetoacetyl-ACP with concomitant loss of COj (Fig. 2). FabH also possesses acetyl-CoA ACP transacylase activity, and for many years it was thought that acetyl-ACP was the actual primer. However, acetyl-ACP appears to be a product of a side reaction, and the role, if any, played by this intermediate in the pathway is unknown. 

Four enzymes participate in each iterative cycle of chain elongation (Fig. 3). The acetoacyl-ACP formed from the initiating FabH condensation is reduced by an NADPH-dependent P-ketoacyl-ACP reductase (fabG), and a water molecule is then removed by a P-hydroxyacyl-ACP dehydrase (fabA otfabZ). The last step is catalyzed by enoyl-ACP reductase (fabl or fabK) to form a saturated acyl-ACP, which serves as the substrate for another condensation reaction or when the chain length reaches 16-18 carbons is utilized for membrane phospholipid synthesis. p-Ketoacyl-ACP synthase I or II (fabB or fabF) initiates additional... 

Fig. 3. Cycles of fatty acyl chain elongation. All intermediates in fatty acid synthesis are shuttled through the cytosol as thioesters of the acyl carrier protein (ACP). (1) P-Ketoacyl-ACP reductase (FabG), (2) P-hydroxyacyl-ACP dehyrase (FabA or FabZ), (3) trani-2-enoyl-ACP reductase I (FabI), (4) P-ketoacyl-ACP synthase I or II (FabB or FabF).

Fig. 4. Branch point in unsaturated fatty acid synthesis. (1) FabA catalyzes the inter-conversion of P-hydroxyde-canoyl-ACP, trans-2-decenoyl-ACP, and ds-3-decenoyl-ACP. (2) rrans-2-Decenoyl-ACP is a substrate for enoyl-ACP reductase (FabI), while (3) cis-3-decenoyl-ACP is elongated by P-ketoacyl-ACP synthase 1 (FabB). Competition between Fabl and FabB is partly responsible for the ratio of saturated to unsaturated fatty acids.

Despite the presence of acetyl-CoA ACP acyltransferase activity in plant fatty acid synthase preparations, acetyl-ACP does not appear to play a major role in plant fatty acid synthesis (J. Jaworski, 1993). Instead, the first condensation takes place between acetyl-CoA and malonyl-ACP. This reaction is catalyzed by P-ketoacyl-ACP synthase III, one of three ketoacyl synthases in plant systems (Fig. 2). The acetoacetyl-ACP product then undergoes the standard reduction-dehydration-reduction sequence to produce 4 0-ACP, the initial substrate of ketoacyl-ACP synthase I. KAS I is responsible for the condensations in each elongation cycle up through that producing 16 0-ACP. The third ketoacyl synthase, KAS II, is dedicated to the final plastidial elongation, that of 16 0-ACP to 18 0-ACP. 

The polyketide synthesis chemically and biochemically resembles that of fatty acids. The reaction of fatty acid synthesis is inhibited by the fungal product ceru-lenin [9]. It inhibits all known types of fatty acid synthases, both multifunctional enzyme complex and unassociated enzyme from different sources like that of some bacteria, yeast, plants, and mammalians [10]. Cerulenin also blocks synthesis of polyketides in a wide variety of organisms, including actinomycetes, fungi, and plants [11, 12]. The inhibition of fatty acid synthesis by cerulenin is based on binding to the cysteine residue in the condensation reaction domain [13]. Synthesis of both polyketide and fatty acids is initiated by a Claisen condensation reaction between a starter carboxylic acid and a dicarboxylic acid such as malonic or methylmalonic acid. An example of this type of synthesis is shown in Fig. 1. An acetate and malonate as enzyme-linked thioesters are used as starter and extender, respectively. The starter unit is linked through a thioester linkage to the cysteine residue in the active site of the enzymatic unit, p-ketoacyl ACP synthase (KS), which catalyzes the condensation reaction. On the other hand, the extender... 

Formation of a fully reduced saturated carbon chain is a three-step process requiring three distinct enzymatic functions. The first step is ketoreduction (P-ketoacyl ACP reductase KR) to produce the secondary alcohol residue in that an electron is supplied by NADPH to the carbonyl group followed by protonation. The second step is dehydration (dehydratase DH) to lead to the a,P unsaturated acyl group. The final step is enoyl reduction (enoyl reductase ER), which employs NADPH as an electron donor and proton to result in the formation of a methylene function at the P-carbon. After the reduction steps are completed, the generated acyl chain enters the KS domain and is equivalent to the starter for the next cycle of the reaction to condense with the next extender unit. 

Fig. 5. Predicted domain organization and biosynthetic intermediates of the erythromycin synthase. Each circle represents an enzymatic domain as follows ACP, acyl carrier protein AT, acyl-transferase DH, dehydratase ER, P-ketoacyl-ACP enoyl reductase KR, [3-ketoacyl-ACP reductase KS, p-ketoacyl-ACP synthase TE, thioesterase. Zero indicates dysfunctional domain.

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

Figure 21-11 Catalytic domains within three polypeptide chains of the modular polyketide sjmthase that forms 6-deoxyerythronolide B, the aglycone of the widely used antibiotic erythromycin. The domains are labeled as for fatty acid synthases AT, acyltransferase ACP, acyl carrier protein KS, P-ketoacyl-ACP synthase KR, ketoreductase DH, dehydrase ER, enoylreductase TE, thioesterase. After Pieper et Courtesy of Chaitan Khosla.

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