0-Ketoacyl synthase

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.

The entire sequence is described in exquisite detail by Smith et al. (2003 see Figure 2.5). The first step is the sequential transfer of the primer, usually acetyl-CoA, to the serine residue of the acyl transferase, then to the ACP, and finally to (3-ketoacyl synthase. The chain extender substrate, usually malonyl-CoA, is transferred via the serine residue of the acyl transferase to ACP. Condensation is accomplished by (3-ketoacylsynthase, aided by the energetically-favourable decarboxylation of the malonyl residue,... 

Animal FASs are functional dimers [76]. While /3-ketoacyl synthase requires dimer formation for activity [77], catalysis of the remaining FAS reactions is carried out by the monomeric enzyme. This behavior is reminiscent of yeast fatty acid synthase, where the -ketoacyl synthase and ACP from different subunits also contribute to the same active site. Electron microscopy and small angle scattering experiments have further defined the structure of the functional complex [34,78]. The overall shape of the molecule, as visualized by electron microscopy, is two side by side cylinders with dimensions of 160x146 x 73 A [34]. 

KS = /3-Ketoacyl synthase MT = malonyl transacylase AT = acetyl transacylase DH = dehydratase ER = enoyl reductase KR = /3-ketoacyl reductase ACP = acyl carrier site TE = thioesterase. [Reproduced with permission from S. J. Wakil, J. K. Stoops, and V. C. Joshi, Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 52, 537 (1983). 1983 by Annual Reviews Inc.]... 

Inherently, the decarboxylation of p-keto acids and malonic acids (1) proceeds very smoothly, as the resulting product bearing anion adjacent to carbonyl group stabilizes as its enolate form (2) [Eq. (1)]. Enzyme-mediated reaction sometimes utilizes this facilitated decarboxylation. Indeed, isocitric acid (3) was oxidized to the corresponding keto acid, which subsequently decarboxylated to a-ketoglutaiic acid (4) by means of isocitrate dehydrogenase (EC 1.1.1.41) [Eq. (2)]. Another example is observed in the formation of acetoacetyl-CoA (5), which occupies the first step of fatty acid biosynthesis. A p-keto carboxylate 6, derived from the acetylation of malonyl-CoA with acetyl-CoA, decarbox-ylates to 5 by the action of 3-ketoacyl synthase [Eq. (3)]. 

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys— SH, cysteine thiol.) The— SH of the 4 -phosphopantetheine of one monomer is in close proximity to the— SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a "head-to-tail" arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

Each CHS monomer consists of two structural domains (Fig. 12.5, left). The upper domain exhibits the a-p-a-p-a pseudo-symmetric motif observed in fatty acid P-ketoacyl synthases (KASs) (Fig. 12.5, right).20 Both CHS and KAS use a cysteine as a nucleophile in the condensation reaction, and shuttle reaction intermediates via CoA thioester-linked molecules or ACPs, respectively. The conserved architecture of the upper domain maintains the three-dimensional position of the catalytic residues of each enzyme Cysl64, His303, and Asn336 in CHS correspond to a Cys, His, and His in KAS I and II. 

Figure 12.5 A. Comparison of the CHS monomer (left) and P-ketoacyl synthase monomer (right). The structurally conserved secondary structure of each monomer s upper domain is colored in blue (a-helix) and gold (P-strand). Portions of each protein monomer forming the dimer interface are colored purple. The side-chains of the catalytic residues of CHS (Cysl64, His303, Asn336) and P-ketoacyl synthase (Cysl63, His303, His340) are shown. B. Sequence conservation of the catalytic residues of CHS, 2-PS, p-ketoacyl synthase (FAS II), and the ketosynthase modules of 6-deoxyerythronolide B synthase (DEBS), actinorhodin synthase (ActI) and tetracenomycin synthase (TcmK). The catalytic residues are in red.

The fatty acid synthase protein is known to contain an acyl carrier protein (ACP) binding site, and also an active-site cysteine residue in the P-ketoacyl synthase domain. Acetyl and malonyl gronps are successively transferred from coenzyme A esters and attached to the thiol groups of Cys and ACP. 

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).

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). 

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 activities involved in yeast fatty acid biosynthesis are covalently linked as separate domains of two multifunctional polypeptides, a and p, encoded by the fas2 and fasl genes, respectively (Fig. 2) [57,58]. The functionalities associated with the 220 kDa a subunit include -ketoacyl synthase activity, -ketoacyl reductase activity, and an AGP domain which bears a phosphopantetheinylated serine. The 208 kDa -subunit has acetyl and malonyl CoA transacylase, palmi-toyl transferase, -hydroxyacyl-enzyme dehydratase, and enoyl acyl-enzyme reductase activities. The two subunits can be readily dissociated, and the individual activities maybe measured [57]. 

The proximity of the -ketoacyl synthase and phosphopantetheine thiols was confirmed in studies using the bifunctional reagent l,3-dibromo-2-propanone, where the two thiols could be effectively cross-linked by the 5 A-long reagent [61,62]. This reagent cross-linked the two reactive thiols in such a way that two a subunits were concomitantly cross-linked. This important finding is the basis for the conclusion that the -ketoacyl synthase/AGP active site is formed from residues derived from two different a subimits. 

Following loading of acetyl and malonyl groups onto the P subunit of the enzyme, additional intramolecular transfers must occur to prepare the substrates for the decarboxylative condensation reaction which is catalyzed by the -ketoacyl synthase domain of the a subunit. The end result of these transfers is the thio-esterification of malonate by the phosphopantetheine thiol and of acetate by Cys-1305(a) of the -keto synthase active site. This cysteine has been shown to have a dramatically lowered pK (<5), which would encourage its reactivity [65]. 

Decarboxylative condensation of the malonyl-ACP onto the -ketosynthase-bound growing acyl chain is likely to be analogous to the corresponding reaction catalyzed by the E. coli -ketoacyl synthase. Once formed, the acetoacetyl derivative remains attached to the phosphopantetheine cofactor during subsequent steps of ketoreduction, dehydration, and enoyl reduction, before the growing fatty acid is transferred to the Cys-1305 thiol in preparation for another round of elongation. 

Elegant experiments, which capitalized on the ability of iodoacetamide to specifically alkylate the active site cysteine of the -ketoacyl synthase, were performed, which definitively proved the capability of the yeast FAS in decar-boxylating malonyl CoA [75]. Following alkylation, FAS activity is abolished however, the enzyme still transacylates malonyl CoA to the phosphopantetheine thiol, where it is decarboxylated before being transferred back to CoA by the transacylase prior to its release as acetyl CoA. 

The seven activities of animal FASs are encoded as separate domains of a single 250 kDa polypeptide (Fig. 2) [30, 31]. These include a -ketoacyl synthase, malonyl/acetyl transferase, -ketoreductase, dehydratase, enoyl reductase, and an ACP domain with a phosphopantetheinylated serine. In addition to these activities, the animal FAS also includes a thioesterase domain which cleaves the product fatty acid from the enzyme. Proteolytic mapping of the polypeptide and genetic analysis have defined the location of the various domains in the primary sequence [30,31]. 

Gel filtration of purified 6-MSAS indicates that it is a 750 kDa homotetramer [120]. When treated with 1,3-dibromopropanone (DBP), 6-MSAS behaves similarly to animal FASs and inactivation occurs concomitantly with its cross-linking to a covalent homodimer. By analogy to the well-characterized DBP cross-linked yeast and animal FASs [61,62,79,80], DBP is suggested to cross-link active site sulfhydryl residues of the -ketoacyl synthase cysteine and the AGP pantetheine. This notion is supported by the observation that preincubation with either acetyl or malonyl CoA precludes cross-linking. Thus, the functional significance of the tetrameric assembly of 6-MSAS remains a mystery. 

FATTY ACID CHAIN EXTENSION CYCLE KS Ketoacyl Synthase ACP = Acyl Carrier Protein KR Ketoreductaso DH =... 

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. 

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