P-ketoacyl synthases

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).

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. 

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.

Several catalytic elements are required for the biosynthetic process acyltransferases that load the primer and extender substrates onto the FAS complex a posttranslationally phosphopantetheinylated acyl carrier protein (ACP) that translocates the various thioester intermediates between catalytic sites a P-ketoacyl synthase (KS) that performs the condensation reaction the p-ketoacylreductase, dehydrase, and enoylreductase enzymes that are responsible for the beta-carbon-processing reactions and a chain-terminating enzyme... 

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... 

The FA elongation stage is catalyzed by P-ketoacyl synthase III (KAS III), a single polypeptide enzyme with several catalytic sites, which allows the enzyme to promote different reactions during this stage (Voelker Kinney, 2001). The condensation of malonyl-ACP and acetyl-CoA to yield 3-ketobutyryl-ACP and CO is the first reaction. The second reaction is the reduction of 3-ketobutyl-ACP to 3-hydroxylacyl-... 

Type I fatty acid synthase. Enzyme activities are arrtmged as a series of globular domains in a single multifunctional protein, 4-5 x 10, consisting of two identical subunits, each of M, 1.8-2.5 x lO , i. e. oj structure. P-Ketoacyl synthase activity is only present in the dimer, since this activity requires juxtaposition of two thiols, one from each subunit. The enoyl reductase does not use a flavin coenzyme (NADPH is used directly for the reduction). Termination is by hydrolysis (thioesterase), and the products are chiefly palmitate with some stearate. 

In bacteria and plants, elongation occurs by continuation of the reactions of F.a.b. beyond Cie, employing synthases of different specificity. 1 vo p-ketoacyl synthases have been isolated from plants, the first producing primarily palmitate, while product distribution shifts primarily to stearate in the presence of the second synthase. Unsaturated acids are elongated in the same way as saturated acids. 

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