P-Ketoacyl-ACP reductase

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. 

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).

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. 

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. 

The studies of the individual enzymes of fatty acid synthesis in higher plants has shown that the two reductive steps, p-ketoacyl ACP reductase and enoyl ACP reductase have different cofactor requirements. As a result the synthesis of fatty acids depends on the availability of both NADH and NADPH. While the provision of NADPH can be attributed to the photosynthetic reactions, the source of NADH in the chloroplast is less certain. Takahama etal (8) have demonstrated that the content of NADPH in the chloroplast is influenced by illumination as expected, but there is no such fluctuation of the oxidation state of NAD/NADH. The production of NADH to be utilized in fatty acid synthesis would therefore appear to depend on dark reactions. One possibility would be by the action of pyruvate dehydrogenase, which would generate not only the NADH required for reduction in fatty acid synthesis but also the precursor acetyl CoA. 

Klein, B., Pawlowski, K., Horicke-Grandpierre, C., Schell, J. and Topfer, R. (1992a) Isolation and characterization of a cDNA from Cuphea lanceoluta encoding a p-ketoacyl-ACP reductase. Mol. Gen. Genet. 233, 122-128. 

Figure 4 (A) Coordinated interaction of members of the condensing (KAS) enzyme family results in biosynthesis of fatty acids in E. coli. The genes encoding each KAS as well as major destinations of the fatty acyl products are shown. Lipoic acid is a precursor of the coenzyme lipoamide. Lipid A consists of p-hydroxymyristate linked to saccharides in the cell membrane. PL are the membrane phospholipids. (B) How do KASes interact with one another and the other members of the FAS complex ACP, acyl carrier protein KR, p-ketoacyl-ACP reductase DH, P-hydroxyacyl-ACP dehydrase ER, enoylacyl-ACP reductase MAL TR, malonyl-CoA ACP transacylase TE, thioesterase AC TR acetyl-CoA ACP transacylase whose contribution to fatty acid synthesis is uncertain since the discovery and characterization of KAS III [33,38].

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