B-Ketoacyl-ACP-Reductase

HYDROXYSTEROID DEHYDROGENASES ISOCITRATE DEHYDROGENASE B-KETOACYL-ACP REDUCTASE... 

Partial separation of 6-ketoacyl-ACP synthetase, 6-ketoacyl-ACP reductase, acetyl CoA ACP transacylase and malonyl-CoAiACP transacylase was achieved from barley chloroplasts . From avocado fruit, Caughey and Kekwlck purified the B-ketoacyl-ACP reductase and malonyl-CoA. ACP acyltransferase to homogeneity and also purified the enoyl-ACP reductase. However, the most thorough study was that by Shlmakata and Stumpf mainly with spinach leaves. Purifications of acetyl CoArACP transacylase, B-ketoacyl-ACP synthetase B-ketoacyl-ACP synthetase II, B-ketoacyl-ACP reductase... 

A cDNA clone has been isolated from a Brassica napus var. Jet Neuf seed library, that encodes B-ketoacyl-[ACP] reductase (3-oxoacyl-ACP reductase) (I3KR) [1]. 

Fig. 4. X-ray determined protein crystal structures of multienzyme ensembly lines, (a) Mammalian fatty acid synthase at 4.5 A resolution (PDB 2cf2). Domain organization A starter substrate, acetyl-CoA or malonyl-CoA, gets loaded onto the acyl-carrler protein (ACP/absent in the structure) via the malonyl-CoA-/acetyl-CoA-ACP transacylase (MAT). Then, the ketoacyl synthase (KS) catalyzes a decarboxylative condensation reaction and forms the B-ketoacyl-ACP. This is followed from a reduction reaction catalyzed by the B-ketoacyl reductase (KR). Subsequently, the Intermediate gets dehydrated by a dehydratase (DH) and additionally reduced by a B-enoyl reductase (ER). The product gets released from the ACP by a thloesterase (absent in the structure), (b) Module 3 of 6-deoxyerthronolide B synthase at 2.6 A resolution (PDB 2qo3) bound to the inhibitor cerulin. The ketosynthase (KS) - acyltransferase (AT) di-domain is part of the large homodimeric polypeptide involved in biosynthesis of erythromycin from Saccharopolyspora erythraea...

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. 

After conversion to acetyl-AGP and malonyl-AGP, two carbons of the malonyl-AGP are introduced via the condensing enzyme, 6-ketoacyl-ACP synthetase. Loss of the malonyl carboxyl drives the reaction and in the first step of the sequence acetoacetyl-AGP is formed. The B-ketoacyl-ACP is then reduced to B-hydroxyacyl-AGP by NADPH and the enzyme B-ketoacyl-AGP reductase. The hydroxy acid is dehydrated to form a trans-2.3-enov1-ACP which can be reduced by NADH or NAOPH to the saturated AGP derivative (butyrate in the first series of steps). Gondensation with malony 1-ACP is then repeated and the cycle continues to produce acyl-ACP derivatives with two additional carbon atoms until palmitoy1-ACP results. A second B-ketoacyl-ACP synthetase accomplishes addition of two more malonyl carbon atoms to allow the formation of stearoyl-ACP. The B-ketoacyl-ACP synthetase has been shown to be a separate enzyme since it is more easily inhibited by arsenite and is less sensitive to the antibiotic, cerulenin, than the B-ketoacyl-ACP synthetase forming C to C g keto acids. 

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].

Fig. 2. Metabolic pathways for PHA biosyntheis in fad mutant E. coli strains used in this study. Enoyl-CoA hydratase, epimerase, and 3-ketoacyl-CoA or ACP reductase have been suggested to supply PHA precursors from inhibited b-oxidation pathway. The crosses indicate inactivation of corresponding enzymes. The question mark represents uncharacterized enzyme. Enzymes involved in the metabolic pathways shown have been described previously FabG (21,32), YfcX (24,33), MaoC (34), PhaA (36), and PhaB (36).

FIGURE 19.4 Modular organization of the six modules (I—VI) of 6-deoxyerythronolide B synthase (DEBS) enzyme as derived from Saccharopolyspora erythraea. Enzyme activities are acyltransferases (AT), acyl carrier proteins (ACP), fi-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 (70), 375 379 383... 

Fig. 2.3 Biosynthesis pathway of A P(3HB) B P(3HB-co-3HV) C P(3HB-co-3HHx) via fatty acid /S-oxidation and D P(3HB-co-3HHx) via fatty acid de novo synthesis. PhaA, f -ketothiolase PhaB, NADPH dependent acetoacetyl-CoA reductase PhaC, PHA synthase PhaG, 3-hydroxyl-ACP-CoA transferase PhaJ, (J )-enoyl-CoA hydratase FabG, 3-ketoacyl-CoA reductase (Sudesh et al. 2000)...

Figure 4 (a) The aromatic polyketide biosynthetic cycle (ACP, acyl carrier protein KS, P-ketoacyl synthase KR, P-ketoacyl reductase DH, dehydratase ER, enoyl reductase), (b) The complex polyketide biosynthetic cycle. 

Figure 1. Proposed pathway for soraphen A biosynthesis. ACP, acyl carrier protein domain AT, acyl transferase DH, dehydratase ER, enoyl reductase KR, ketoacyl reductase KS, ketoacyl synthase TE, thioesterase. The inactive DH in module 8 is shown as a square. Adapted with permission from reference (9). Copyright 2002 Elsevier Science B. V.

Fig. 7.1 The FASN Enzyme. A. The FASN polypeptide comprises seven functional domains the ketoacyl synthase (KS), malonyl acetyl transferase (MAT), P-hydroxyacyl dehydratase (DH), enoyl reductase (ER), P-ketoacyl reductase (KR), the acyl carrier protein (ACP), and thioesterase (TE) domains. B. The FASN reaction mechanism. The MAT domain of the enzyme binds malonyl-CoA and acetyl-CoA, while the KS domain acts to condense the growing acyl chain. The resulting P-ketoacyl moiety is then reduced in steps by the KR, DH, and ER to a saturated acyl intermediate. This process is repeated in seven cycles, after which, the TE domain releases the sixteen carbon fatty acid palmitate...

B.N. Wu, Y.M. Zhang, Z. lie, C.O. Rock, Key residues responsible for acyl carrier protein (ACP) and beta-ketoacyl-acyl carrier protein reductase (FabG) interaction. J. Biol. Chem. 278, 52935-52943 (2004)... 

Figure 3.7 Model of intermolecular fatty acid synthetase mechanism in the a2 2 protomer of yeast. A, acetyl transferase E, enoyl reductase D, dehydratase P, palmitoyl transferase M, malonyl transferase C, 5-ketoacyl synthase R. )5-ketoacyl reductase ACP, acyl carrier protein. Dotted lines and arrows delineate the route taken by intermediates when sequentially processed on different FAS domains. Numbers indicate the reaction sequence. Catalytically active dohnains, at a specific moment, are marked by bold lines. Shaded areas on E and P domains potentially interact by hydrophobic attraction in the presence of palmitate (b). On the protomer depicted in (a) fatty acyl chain elongation occurs in one half of the a2 2 protomer. In (b) chain termination is induced by hydrophobic interaction between E> bound palmitate and P. Subsequently, palmitate Is transferred to Its O-ester binding site on P. Inactivation of the left half of simultaneously activates its right half (b). Redrawn from Schweizer (1984) with permission of the author and Elsevier Science Publishers, BV. From Fatty Acid Metabolism and its Regulation (1984) (ed. S. Numa), p. 73, Figure 7.

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