3-Ketoacyl-acyl carrier protein reductase

Shimakata, T., Stumpf, P.K. Purification and characterizations of / -ketoacyl-[acyl-carrier-protein] reductase, / -hydroxyacyl-[acylcarrier-protein] dehydrase, and enoyl-... 

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)... 

The 3-keto reductase step is equivalent to that found in fatty acid synthesis, although it occurs late (and only once) in this process. Indeed, thiol-bound acetoacetate proved inactive as a substrate for the aromatic complex, whereas it was reduced by fatty acid synthetase (Dimrothe/a/., 1972). Thus, the carbonyl group adjacent to the terminal methyl position is not susceptible to reduction by the aromatic synthetase, despite the apparent presence of a suitable reductase, possibly because it is held on the enzyme surface in an inappropriate enolic configuration (Packter, 1973). If so, it may not prove acceptable, since the 3-ketoacyl-acyl carrier protein (ACP) reductase from Escherichia coli only accepts keto substrates and does not react with or bind to the enol form of 3-ketoacyl derivatives (Schulz and Wakil, 1971). 

Although it was known that the intermediates of the p-oxidation cycle are channelled towards PHA biosynthesis, only recently the precursor sources were identified. In A. caviae, the p-oxidation intermediate, trans-2-tnoy -CoK is converted to (R)-3-hydroxyacyl-CoA via (R)-specific hydration catalysed by a (R)-specific enoyl-CoA hydratase [122, 123]. Subsequently, Tsuge and co-workers [124] reported the identification of similar enoyl-CoA hydratases in F. aeruginosa. In the latter case, two different enoyl-CoA hydratases with different substrate specificities channelled both SCL and MCL enoyl-CoA towards PHA biosynthesis. In recombinant . coli it was further shown that 3-ketoacyl-CoA intermediates in the p-oxidation cycle can also be channelled towards PHA biosynthesis by a NADPH-dependent 3-ketoacyl-acyl carrier protein (ACP) reductase [125]. A similar pathway was also identified in F. aeruginosa [126]. In addition, it was also reported that the acetoacetyl-CoA reductase (PhaB) of R. eutropha can also carry out the conversion of 3-ketoacyl-CoA intermediates in Pathway II to the corresponding (R)-3-hydroxyacyl-CoA in E. coli [127]. The results clearly indicate that several channelling pathways are available to supply substrates from p-oxidation cycle to the PHA synthase. This explains why it was not possible to obtain mutants that completely lack PHA accumulation ability, unless the mutation occurred in the PHA synthase gene [128]. 

Figure 3.8 One complete cycle and the first step in the next cycle of the events during the synthesis of fatty acids. ACP = acyl carrier protein, a complex of six enzymes i.e. acetyl CoA-ACP transacetylase (AT) malonyl CoA-ACP transferase (MT) /3-keto-ACP synthase (KS) /J-ketoacyl-ACP reductase (KR) / - hydroxyacyl-ACP-dehydrase (HD) enoyl-ACP reductase (ER).

ACP acyl carrier protein AT acyltransferase DH dehydratase ER enoyl reductase KR p-ketoacyl reductase KS p-ketoacyl synthase TE thioesterase... 

Figure 3 The fatty acid biosynthetic cycle (ACP, acyl carrier protein KS, P-ketoacyl synthase KR, P-ketoacyl reductase DH, dehydratase ER, enoyl reductase TE, thioes-terase).

Figure 5 Domain organization of the erythromycin polyketide synthase. Putative domains are represented as circles and the structural residues are ignored. Each module incorporates the essential KS, AT, and ACP domains, while all but one include optional reductive activities. AT, acyltransferase ACP, acyl carrier protein KS, (3-ketoacyl synthase KR, P-ketoacyl reductase DH, dehydratase ER, enoyl reductase TE, thioesterase.

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... 

The )9-ketoacyl-synthases/acyltransferases (KS/ AT) in each module effect the chain elongation by methyl-malonyl-coenzyme A units catalyzing a Claisen e.ster condensation followed by decarboxylation (Scheme 2). Subsequent domains are module-specific ketoreductases (KR), dehydratases (DH) or enoyl-reductases (ER), which regulate the functionalization of the newly prepared fi-oxoesters. The stepwise growing chain is picked up by an acyl-carrier protein (ACP). 

Figure 22.23. Schematic Representation of Animal Fatty Acid Synthase. Each of the identical chains in the dimer contains three domains. Domain 1 (blue) contains acetyl transferase (AT), malonyl transferase (MT), and condensing enzyme (CE). Domain 2 (yellow) contains acyl carrier protein (ACP), P-ketoacyl reductase (KR), dehydratase (DH), and enoyl reductase (ER). Domain 3 (red) contains thioesterase (TE). The flexible phosphopantetheinyl group (green) carries the fatty acyl chain from one catalytic site on a chain to another, as well as between chains in the dimer. [After Y. Tsukamoto, H. Wong, J. S. Mattick, and S. J. Wakil. J. Biol. Chem. 258(1983) 15312.]...

Fig. 2. Predicted domain organisation of the DEBS Proteins. Ketoacyl Synthase (KS) Acyl Transferase (AT) Dehydratase (DH) Enoyl Reductase (ER) Keto Reductase (KR) Acyl Carrier Protein (ACP) Thioesterase (TE). Each domain is represented by a box with coded shading whose length is proportional to the size of the domain (KR) indicates an inactive KR domain. The ruler indicates the residue number within the primary structure of the constituent proteins. Linker regions are shown in proportion...

ACP = acyl carrier protein KS = p-ketoacyl synthase KR = p-ketoacyl reductase ER = enoyl reductase DH = dehydratase TE = thioesterase... 

The answer is e. (Murray, pp 230-267. Scriver, pp 2297-2326. Sack, pp 121-138. Wilson, pp 287-320.) The fatty acid synthase complex of mammals is composed of two identical subunits. Each of the subunits is a multienzyme complex of seven enzymes and the acyl carrier protein component. All the components are covalently linked together thus, all the components are on a single polypeptide chain, which functions in the presence of another identical polypeptide chain. Each cycle of fatty acid synthesis employs the acyl carrier protein and six enzymes acetyl transferase, malonyl transferase, p-ketoacyl synthase, p-ketoacyl reductase, dehydratase, and enoyl reductase. When the final fatty acid length is reached (usually C16), thioesterase hydrolyzes the fatty acid off of the synthase complex. 

Abbreviations FASN, fatty acid synthase ACC, acetyl-CoA-carboxylase ACL, ATP-citrate lyase NADPH, nicotinamide adenine dinucleotide phosphate MAT, malonyl acetyl transferases KS, ketoacyl synthase KR, p-ketoacyl reductase DH, p-hydroxyacyl dehydratase ER, enoyl reductase TE, thioesterase ACP, acyl carrier protein VLCFA, very long chain fatty acids ELOVL, elongation of very long chain fatty acids SCDl, stearoyl-CoA desaturase-1 AMPK, AMP-activated kinase ME, malic enzyme FASKOL, liver-specific deletion of FAS PPARa, Peroxisome Proliferator-Activating Receptor alpha HMG-CoA, 3-hydroxy-3-methyl-glutaryl-CoA SREBP, sterol response element binding protein SIP, site-one protease S2P, site-two... 

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...

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. 3. Generic reaction sequence for the FASs. ACP, acyl carrier protein AT, acetyltransferase MT, malonyl transferase KS, P-ketoacyl synthase KR, P-ketoacyl reductase DH, dehydrase ER, enoyl reductase TE, thioesterase FT, palmitoyl transferase. In the animal FAS the acetyl and malonyl loading reactions are catalyzed by the same acyl transferase and the chain-termination reaction is catalyzed by a thioesterase. In the fungal FAS, the malonyl loading and palmitoyl unloading reactions are catalyzed by the same acyl transferase. Stereochemical analyses in the laboratories of Comforth and Hammes established that in both animal and fungal FASs the KS-catalyzed condensation reaction proceeds with inversion of configuration at the malonyl C2 position, followed by KR-catalyzed reduction of the 3-keto moiety to the 3R alcohol by transfer of the pro-4S hydride from NADPH, and DH-catalyzed dehydration to a trans-enoyl moiety by the syn elimination of the 2S hydrogen and the 3/f hydroxyl as water. However, the stereochemistry of the final reduction reaction catalyzed by ER domain proceeds with different stereochemistry. The animal FAS transfers the pro-4R hydride of NADPH to the pro-3/f position with simultaneous addition of a solvent proton to the pro-2S position, whereas the fungal FAS takes the pro-4S hydride of NADPH into the pro-3S position and the solvent proton is incorporated at the pro-25 position.

To carry out these processes, the fatty acid synthase required a set of catalytic activities that are responsible for the respective step of a set of the cycle reactions. Acyltransferase (AT) transfers both the acetyl starter unit and malonyl chain-extender units from the respective coenzyme A esters to the appropriate thiol group of the synthase. P-Ketoacyl synthase and acyl carrier protein are responsible for chain elongation. P-Ketoreductase, dehydratase, and enoyl reductase are for... 

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