Fatty acids synthase sequence

Acyl carrier protein, fatty acid synthase sequence, 45-46 Acyl carrier protein derivatives, desaturation, 13,l4f Acyl lipids, formation, 47-48 Aglycone skeletons saponins, 288f Solanum glycoalkaloids, 288f... 

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.

Fatty acid synthesis is catalysed in animals by the enzyme fatty acid synthase, which is a multifunctional protein containing all of the catalytic activities required. Bearing in mind the necessity to provide a specific binding site for the various substrates involved, and then the fairly complex sequence of reactions carried out, it raises the question of just how it is possible for this process to be achieved at the enzymic level. Nature has devised an elaborate but satisfyingly simple answer to this problem. 

FIGURE 21-5 Sequence of events during synthesis of a fatty acid. The fatty acid synthase complex is shown schematically. Each segment of the disk represents one of the six enzymatic activities of the complex. At the center is acyl carrier protein (ACP), with its phosphopantetheine arm ending in an —SH. The enzyme shown in blue is the one that will act in the next step. As in Figure 21-3, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as C02 is shaded green. Steps (T) to (7) are described in the text. 

In higher animals as well as in My cobacterium,207 yeast,208 and Euglena, the fatty acid synthase consists of only one or two multifunctional proteins. The synthase from animal tissues has seven catalytic activities in a single 263-kDa 2500-residue protein 209 The protein consists of a series of domains that contain the various catalytic activities needed for the entire synthetic sequence. One domain contains an ACP-like site with a bound 4 -phosphopantetheine as well as a cysteine side chain in the second acylation site. This synthase produces free fatty acids, principally the C16 palmitate. The final step is cleavage of the acyl-CoA by a thioesterase, one of the seven enzymatic activities of the synthase. See Chapter 21 for further discussion. 

A detailed study of amino acid sequences and mechanistic similarities in various polyketide synthase (PKS) enzymes has led to two main types being distinguished. Type I enzymes consist of one or more large multifunctional proteins that possess a distinct active site for every enzyme-catalysed step. On the other hand, Type II enzymes are multienzyme complexes that carry out a single set of repeating activities. Like fatty acid synthases, PKSs catalyse the condensation of coenzyme A esters of simple carboxylic acids. However, the variability at each step in... 

We were also able to use FAB mass spectrometry to determine the amino acid sequence around the active site serine in the acyl transference domain of rabbit mammary fatty acid synthase.6 The synthase was labelled in the acyl transferase domain(s) by the formation of O-ester intermediates after incubation with [" " C]-acetyl- or malonyl-CoA (Fig. 2A). The modified protein was then digested with elastase (Fig. 2B), and radioactive material isolated via successive purification steps on Sephadex G-50 and reverse phase HPLC. The isolated peptides were then sequenced by FAB MS. The data summarized in Table II established the sequences of both the acetyl and malonyl hexapeptides to be N-acyl-Ser-leu-Gly-Glu-Val-Ala. 

Figure 2.5. Reaction sequence for the biosynthesis of fatty acids de novo by the animal FAS. The condensation reaction proceeds with stereochemical inversion of the malonyl C-2, the (3-ketoacyl moiety is reduced by NADPH to D-(3 hydroxyacyl moiety, which then is dehydrated to a trans-enoyl moiety finally, the enoyl moiety is reduced to a saturated acyl moiety by NADPH, with the simultaneous addition of a solvent proton. The two C atoms at the methyl end of the fatty acid are derived from acetyl-CoA, the remainder from malonyl-CoA. The entire series of reactions takes approximately 1 second. PSH, phosphopantetheine. (Reprinted from Prog, in Lipid Res., vol. 42, S. Smith, A. Witkowski and A.K. Joshi, Stuctural and functional organization of the animal fatty acid synthase, pp. 289-317, copyright (2003), with permission from Elsevier).

Polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases are a structurally and mechanistically related class of enzymes that catalyze the synthesis of biopolymers in the absence of a nucleic acid or other template. These enzymes utilize the common mechanistic feature of activating monomers for condensation via covalently-bound thioesters of phosphopantetheine prosthetic groups. The information for the sequence and length of the resulting polymer appears to be encoded entirely within the responsible proteins. 

Several architectural paradigms are known for polyketide and fatty acid synthases. While the bacterial enzymes are composed of several monofunctional polypeptides which are used during each cycle of chain elongation, fatty acid and polyketide synthases in higher organisms are multifunctional proteins with an individual set of active sites dedicated to each cycle of condensation and ketoreduction. Peptide synthetases also exhibit a one-to-one correspondence between the enzyme sequence and the structure of the product. Together, these systems represent a unique mechanism for the synthesis of biopolymers in which the template and the catalyst are the same molecule. 

This four-step cycle includes condensation of acetate and malonate to give ketobu-tanoate with subsequent reduction to butanoate in three further steps. These are reduction to the 3R hydroxy acid, dehydration to the 2t acid, and reduction again. Reduction is affected by NADPH and a proton. The process is then repeated to add further two-carbon units until a thioesterase liberates the free acid. This sequence requires a fatty acid synthase, which contains the enzymes needed for each of the four steps viz. p-ketoacyl-ACP synthase, p-ketoacyl-ACP reductase, p-ketoacyl-ACP dehydrase, and enoyl-ACP reductase, respectively. 

B. The synthesis of fatty acids from glucose occurs in the cytosol, except for the mitochondrial reactions in which pyruvate is converted to citrate. Biotin is required for the conversion of pyruvate to oxaloacetate, which combines with acetyl CoA to form citrate. Biotin is also required by acetyl CoA carboxylase. Pantothenic acid is covalently bound to the fatty acid synthase complex as part of a phosphopantetheinyl residue. The growing fatty acid chain is attached to this residue during the sequence of reactions that produce palmitic acid. NADPH, produced by the malic enzyme as well as by the pentose phosphate pathway, provides reducing equivalents. Citrate, not isocitrate, is a key regulatory compound. 

Regulation of metabolic processes can be accomplished by other methods. One is the use of a multienzyme complex (e.g., pyruvate dehydrogenase complex or fatty acid synthase complex) in which various enzymes are organized such that the product of one becomes the substrate for an adjacent enzyme. A single polypeptide chain may contain multiple catalytic centers that carry out a sequence of transformations (e.g., the mammalian fatty acid synthase see Chapter 18). Such multifunctional polypeptides increase catalytic efficiency by abolishing the accumulation of free intermediates and by maintaining a stoichiometry of 1 1 between catalytic centers. 

The reactions of de novo fatty acid biosynthesis are shown in Figure 18-10. They are carried out by two multienzyme systems functioning in sequence. The first is acetyl-CoA carboxylase, which converts acetyl-CoA to malonyl-CoA. The second is fatty acid synthase, which sequentially joins two-carbon units of malonyl-CoA, eventually producing palmitic acid. Both complexes consist of multifunctional subunits. The various catalytic functions can be readily separated in plant cells and prokaryotes, but in yeasts, birds, and mammals, attempts to subdivide catalytic functions lead to loss of activity. Important features of this system are as follows ... 

Despite their enormous structural diversity, polyketide metabolites are related by their common derivation from highly functionalised carbon chains whose assemblies are controlled by multifunctional enzyme complexes, the polyketide synthases (PKSs) which, like the closely related fatty acid synthases, catalyse repetitious sequences of decarboxylative condensation reactions between simple acyl thioesters and malonate, as shown in Fig. 3 [7]. Each condensation is followed by a cycle of modifying reactions ketoreduction, dehydration and enoyl reduction. In contrast to fatty acid biosynthesis where the full cycle of essentially reductive modifications normally follow each condensation reduction, the PKSs can use this sequence in a highly selective and controlled manner to assemble polyketide intermediates with an enormous number of permutations of functionality along the chain. As shown in Fig. 3, the reduction sequence can be largely or entirely omitted to produce the classical polyketide intermediate which bears a carbonyl on every alternate carbon and which normally cyclises to aromatic polyketide metabolites. On the other hand, the reductive sequence can be used fully or partially after each condensation to produce highly functionalised intermediates such as the Reduced polyketide in Fig. 3. Basic questions to be answered are (i) what is the actual polyketide intermediate... 

---