Acetyl-CoA:ACP transacylase

Fatty acid synthesis begins when the substrates, acetyl-CoA and malonyl-CoA, are transferred onto the protein by malonyl-CoA acetyl-CoA-ACP transacylase (MAT, steps 1 and 2 in fig. 18.12a). The numbers in parentheses below the abbreviation of the enzyme in this figure refer to the reactions shown in fig. 18.12. (Whereas E. coli has separate enzymes that catalyze the transfer of acetyl- and malonyl-CoA to ACP, both reactions are catalyzed by the same enzymatic activity (MAT) on the animal fatty acid synthase.) Subsequently, /3-ketobutyryl-ACP and CO2 are formed in a condensation reaction catalyzed by /3-ketoacyl-ACP synthase (KS, step 3 in fig. 18.12a). 

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...

The first reaction, catalyzed by Acetyl-CoA ACP transacylase, transfers an acetyl group from Coenzyme A to the cysteinyl-S on ACPi. 

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. 

Acetyl-CoA + ACP <=> Acetyl-ACP + CoASH (catalyzed by Acetyl-CoA-ACP Transacylase)... 

Acetyl-CoA-ACP Transacylase is an enzyme of fatty acid biosynthesis that catalyzes the transfer of acyl carrier protein (ACP) to acetyl-CoA in the reaction below (Figure 18.24). Acetyl-ACP is the initial two carbon unit onto which the initial malonyl-ACP is added to form / -ketoacyl-ACP. 

ACP is covalently linked to acetyl-CoA and malonyl-CoA by the enzymes acetyl-CoA-ACP transacylase and malonyl-CoA-ACP transacylase, respectively. 

Transfer of the acetyl group of acetyl-CoA to ACP-catalyzed by acetyl-CoA-ACP transacylase ... 

Because the bacterial enzymes are much more readily soluble than the mammalian, the steps of fatty acid synthesis have been elucidated in E. coli. The multiple-enzyme complex has been resolved into at least 7 protein components, 6 of which have specific enzyme activities. The other is referred to as the acyl carrier protein. The bacterial carrier protein (mol wt approximately 16,000) is rich in acidic residues and contains a 4 -phosphopantothenic prosthetic group, which, as we shall see, plays a key role in the binding of the acyl group. The prosthetic group is bound to a serine residue of the carrier protein. The carrier protein acts as an acceptor of the acetyl group attached to the SH group of CoA in a reaction catalyzed by an enzyme, acetyl-CoA-ACP transacylase ... 

Thus, in the reaction the acetyl group is transferred from the SH group of acetyl-CoA to a thioester linkage with the SH group of the 4 -phosphopantothenic moiety. The acetyl-CoA-ACP transacylase has been partially purified although not absolutely specific for acetyl-CoA, it reacts poorly with other CoA derivatives, such as propionyl CoA. An acetyl enzyme intermediate is formed in the course of the reaction. 

The malonyl-coenzyme A then reacts with acyl-carrier protein (ACP), in the presence of malonyl-CoA-ACP transacylase, to give the malonyl-ACP complex. Acetyl-coenzyme A is then coupled with ACP in the presence of acetyl-CoA-ACP transacylase, and this reacts with the malonyl-ACP, the chain length being increased by two carbon atoms to give the butyryl-ACP complex. The reactions involved are shown in Fig. 9.18. 

Although [ C]acetate is a relatively poor precursor for fatty acid synthesis in stromal fractions, fluazifop did inhibit labelling from this compound. By blocking labelling via acetyl-CoA ACP transacylase (with unlabelled acetyl-ACP) or via acetyl-CoA carboxylase (with avidin and unlabelled malonyl-CoA) we were able to show that the fluazifop action was at the level of acetyl-CoA carboxylase. 

The most complete examination of the relative rates for the individual activities listed above, including Cuphea, Scifflower and rapeseeds, suggests acetyl-CoA ACP transacylase and B-ketoacyl-ACP synthetase as rate-limiting. ... 

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... 

In recent years, the molecular organization of the plant fatty acid synthetases has been examined in extracts from various plant tissues, and in all cases was found to be nonassociated and similar to the prokaryotic type of coli (1,2,3). Many of the individual component enzymes have been partially purified Including malonyl-CoAtacyl carrier protein (ACP) trans-acylase (4), 3-ketoacyl-ACP reductase (5,6), E-hydroxylacy1-ACP dehydrase (6), enoyl ACP reductase (5,6) and acetyl-CoA ACP transacylase (ATA) (1). 

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].

THE PURIFICATION OF ACETOACYL CARRIER PROTEIN SYNTHASE FROM AVOCADO AND IDENTIFICATION OF A SEPARATE ACETYL COA ACP TRANSACYLASE ACTIVITY... 

The biosynthesis of fatty acids in plants Is catalysed by a type II, dissociable, fatty acid synthetase (FAS) made up of at least six catalytic polypeptides and a central, acyl carrier protein (ACP). There are three p Ketoacyl-ACP synthetases (KAS) KAS 1, catalysing the initial reaction between acetyl ACP and malonyl ACP KAS 2, catalysing the addition of acetyl ACP to the elongating fatty acid chain and the recently identified KAS 3. A further enzyme implicated in the initiation of FAS is acetyl CoA ACP transacylase (AC AT) [3]. AC AT catalyses the formation of acetyl ACP. KAS 3, however does not utilise acetyl ACP, instead the condensation occurs between malonyl-ACP and acetyl CoA. There has recently been much discussion about the possible roles of KAS 3 and ACAT which appear to have partially overlapping functions KAS 3 activity has been purified to homogeneity from spinach [1] and E. coli [4]. In both the purified enzyme has the ability to load an acetyl group from acetyl CoA, ACAT activity. Purification of ACAT from E. coli has been reported [3], but as this work predates the discovery of KAS 3 it is not clear whether this activity is resolvable from KAS 3. There is still confusion as to whether plants posses distinct ACAT activity since the assays that have been used for both enzymes contain ACP. 

Q jliver BS Slabas AR Acetoacyl-acyl carrier protein synthase from Avocado-purificaticn, characterisation and clear resolution from acetyl CoA ACP transacylase. Plant mol Biol in press. 

Acetyl-CoA ACP transacylase is a thiolactomycin-sensitive enzyme which catalyzes what has often been regarded as the slowest of all the partial reactions. However, the recent observation that in spinach leaves and all other plant tissues examined there is an acetoacetyl-ACP synthetase which bypasses this reaction (and which has much higher activity) sheds doubt on the physiological importance of acetyl-CoA ACP transacylase. The acetoacetyl-ACP synthetase, sometimes called the short-chain condensing enzyme, is cerulenin insensitive and can, therefore, be differentiated easily from the )8-ketoacyl-ACP synthetase I, which is the main condensing enzyme. The latter has been purified recently to homogeneity for the first time. " ... 

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