Acyl transacylase

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

Malonyl-CoA Acyl carrier protein transacylase (MCAT or FabD) catalyzes the transfer of a malonyl group between coenzyme A and acyl carrier proteins that are the privileged transporters of the FASH system. Although essential, this enzyme is present in excess and does not have a regulatory role in the FASH pathway, which might explain the relative paucity of reported inhibitors [1],... 

Selected entries from Methods in Enzymology [vol, page(s)] Assay, 1, 611 3, 935-938 63, 33 separation by HPLC, 72, 45 extraction from tissues, 13, 439 formation of, 1, 486, 518, 585 5, 466 free energy of hydrolysis, 1, 694 substrate for the following enzymes [acetyl-coenzyme A acyl carrier protein transacylase, 14, 50 acetyl-coenzyme A carboxylase, 14, 3, 9 acetyl-coenzyme A synthetase, 13, 375 N-acetyltransferase, 17B, 805 aminoacetone... 

After malonyl-CoA synthesis, the remaining steps in fatty acid synthesis occur on fatty acid synthase, which exists as a multienzyme complex. In the initial reactions acetyl-CoA and malonyl-CoA are transferred onto the protein complex by acetyl-CoA transacylase and malonyl-CoA transacylase (step 1 and step 2 in fig. 18.12a). The acceptor for the acetyl and malonyl groups is acyl carrier protein (ACP). ACP also carries all of the intermediates during fatty acid biosynthesis. The prosthetic group that binds these intermediates is... 

We saw in Section 12.3.1 the use of the cyclodextrins as mimics for transacylases, a well-understood class of enzymes that perform the task of transferring an acyl group from one substrate to another (e.g. from an ester to water). Transacylase chemistry has also been addressed by Cram,5 who used chiral corands, such as 12.11, related to 3.106 bearing thiolate nucleophiles situated above and below the plane of the macrocycle. An acyclic analogue 12.12 was also prepared for comparison. The salient features of 12.11 are shown diagrammatically in Figure 12.4. 

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...

Haydock SF, Aparicio JF, Mobiar I, Schwecke T, Khaw LE, Konig A, Marsden AF, Galloway IS, Staunton J, Leadlay PF (1995) Divergent Sequence Motifs Correlated with the Substrate Specificity of (Methyl) mMalonyl-CoA Acyl Carrier Protein Transacylase Domains in Modular Polyketide Synthases. FEBS Lett 374 246... 

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]. 

Crawford JM, Dancy BCR, Hill EA, Udwary D, Townsend CA. Identification of a starer unit-acyl carrier protein transacylase domain in an iterative type 1 polyketide synthase. Proc. Natl. Acad. Sci. U.S.A. 2006 103 16728-16733. 

Malonyl transacylase is highly specific, whereas acetyl transacylase can transfer acyl groups other than the acetyl unit, though at a much slower rate. Fatty acids with an odd number of carbon atoms are synthesized starting with propionyl ACP, which is formed from propionyl CoA by acetyl transacylase. 

Steps 1-2 of Figure 29.5 Acyl Transfers The starting material for fatty-acid synthesis is the thioester acetyl CoA, the ultimate product of carbohydrate breakdown, as we ll see in Section 29.6. The synthetic pathway begins with several priming reactions, which transport acetyl CoA and convert it into more reactive species. The first priming reaction is a nucleophilic acyl substitution reaction that converts acetyl CoA into acetyd ACP (acyl carrier protein). The reaction is catalyzed by ACP transacylase. 

Three types of subunit proteins constitute such dehydrogenase complexes (1) a TPP-dependent decarboxylase, which converts the a-keto acid to an a-hydroxyalkyi-TPP complex (2) a transacylase core, which contains lipoyl residues that are acylated by the a-hydroxyalkyl-TPP and (3) a flavin adenine dinucleotide (FAD)-dependent dihy-drolipoyi dehydrogenase, which reoxidizes the reduced Hpoyl... 

Burstein and Hunter (1995) observed that THC stimulated the biosynthesis of anandamide in neuroblastoma cells employing either ethanolamine or arachidonic acid as the label. Anandamide bios5mthesis has also been shown to occur in primary cultures of rat brain neurons labelled with [H]-ethanolamine when stimulated with ionomycin, a Ca ionophore (Di Marzo et al. 1994). These authors proposed an alternate model for the biosynthesis of anandamide in which N-arachidonoyl phosphatidyl ethanolamine is cleaved by a phospholipase D activity to yield phosphatidic acid and ararchidonoylethanolamide. This model is based upon extensive studies undertaken by Schmid and collaborators (1990), who have shown that fatty acid ethanolamide formation results from the N-acylation of phosphatidyl ethanolamine by a transacylase to form N-acyl phosphatidylethanolamine. Possibly resulting from postmortem changes, this compound is subsequently hydrolyzed to the fatty acid ethanolamide and the corresponding phosphatide by a phosphodiesterase, phospholipase D. 

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.]... 

Yamashita A, Sugiura T, Waku K. Acyl transferases and transacylases involved in fatty acid remodeling of phosphohpids and metabolism of bioaclive hpids in mammalian cells. J Biochem (Tokyo) 1997 122 1-16. 

Serine carbohydrate esterases and transacylases. The commonest reaction mechanism is the standard serine esterase /protease mechanism, demonstrated paradigmally for chymotrypsin, involving an acyl-enzyme intermediate. The enzyme nucleophile is a serine hydroxyl, which is hydrogen bonded the imidazole of a histidine residue, whose other nitrogen is hydrogen bonded to a buried, but ionised, aspartate residue (Figure 6.28),... 

During the first reaction on fatty acid synthase, acetyl transacylase catalyzes the transfer of the acetyl group from an acetyl-CoA molecule to the SH group of a cysteinyl residue of /J-ketoacyl-ACP synthase. Malonyl-ACP is formed when malonyl transacylase transfers a malonyl group from malonyl-CoA to the SH group of the pantetheine prosthetic group of ACP (reaction 2). Then /J-keto-acyl-ACP synthase catalyzes a condensation reaction (reaction 3) in which ace-toacetyl-ACP is formed (Figure 12.14). 

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