Acyltransferases, modification

These enzymatic activities include three acyltransferases (HtrBl, HtrB2, PagP) and a deacylase (PagL). Depending on the specific PA isolate background, these activities can be classified as inducible (lipid A structures only observed under specific growth conditions that induce modification in these isolates), constitutive (lipid A structures always observed under any growth condition in these... 

Acetylation of antibiotics is a common mechanism of modification. The acyltransferases generally use the metabolically abundant acetyl-CoA as the preferred acyl-donor. The aminoglycoside antibiotic acetyltransferases (AACs), for example, modify important amines that make contact with the target 16 S rRNA (Fig. 9), which results in a loss of positive charge and increased steric bulk that contribute to lowering the affinity of the antibiotics for the rRNA target by 600-fold (26). The AACs are members of the GCN5 superfamily of acyltransferases that include such members as the eukaryotic histone acetyltransferases and serotonin acetyltransferase (27). 

The three CAAX-signaled modifications increase the overall hydrophobicity of the carboxyl terminus of Ras and are necessary to promote Ras membrane association. However, these modifications alone are not sufficient to direct full plasma membrane association and signaling activity. At least two additional motifs exist at the carboxyl termini of Ras proteins, which serve to facilitate plasma membrane association and direct Ras proteins to discrete membrane subdomains. These motifs function as secondary signals and are composed either of a stretch of basic amino acids (K-Ras4B) or of cysteine(s) that are pahni-toylated (H-Ras, N-Ras, K-Ras4A) and positioned immediately upstream of the CAAX motif (28, 30) (see Fig. 3). The addition of a palmitate fatty acid is catalyzed by a protein acyltransferase, which forms a reversible thioester bond between the cysteine and the pahnitoyl group (31, 32). 

The liver synthesizes two enzymes involved in intra-plasmic lipid metabolism hepatic triglyceride lipase (HTL) and lecithin-cholesterol-acyltransferase (LCAT). The liver is further involved in the modification of circulatory lipoproteins as the site of synthesis for cholesterol-ester transfer protein (CETP). Free fatty acids are in general potentially toxic to the liver cell. Therefore they are immobilized by being bound to the intrinsic hepatic fatty acid-binding protein (hFABP) in the cytosol. The activity of this protein is stimulated by oestrogens and inhibited by testosterone. Peripheral lipoprotein lipase (LPL), which is required for the regulation of lipid metabolism, is synthesized in the endothelial cells (mainly in the fatty tissue and musculature). 

Mathur, S.N., Simon, L, Lokesh, B.R., Specter, AA. Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A cholesterol acyltransferase activity. Biochem. Biophys. Acta 1983 751 401-411... 

Wnt proteins are also acylated. The Wnt3a protein is modified by thioester-linked palmitate at a conserved cysteine residue and also by an unsaturated fatty acid, palmitole-ic acid, which is oxyester-linked to a conserved serine residue (R. Takada, 2006). Porcupine (pore), a member of the MBOAT family, is required for the O-acylation of Wnt3a. It is not clear whether pore or another acyltransferase carries out the 5-acyl modification. 

Fig. 5. Pathway depicting how flux through phosphatidylcholine (product of reaction 3) can promote acyl group diversity in plant triacylglycerols. Production of 18 2 (boxed) at the sn-2 position and its transfer to TG is used as a sample modification. Other fatty acid alterations may be substituted. Enzymes 1, glycerol-3-phosphate acyl-CoA acyltransferase and lysophosphatidic acid acyl-CoA acyltransferase 2, phosphatidic acid phosphatase 3, diacylglyceroliCDP-aminoalcohol aminoalcoholphosphotransferase 4, 18 l-desaturase or other fatty acid modifying enzyme 5, phosphlipid diacylglycerol acyltransferase 6, diacylglycerol acyltransferase 7, acyl-CoA phosphatidylcholine acyltransferase or phospholipase plus acyl-CoA synthetase.

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

Zou, J.-T., V. Katavic, E.M. Giblin, et al. 1997. Modification of seed oil content and acyl composition in Brassicaceae by expression of a yeast sn-2 acyltransferase gene. Plant Cell 9 909-923. 

Inhibitors of acyl CoA-cholesterol acyltransferase (ACAT) are currently being Investigated as cholesterol-lowering or antiatherosclerotic agents. In addition to its role in foam cell formation, ACAT also is required for esterification of cholesterol in intestinal mucosal cells and for synthesis of cholesterol esters in hepatic VLDL formation. Thus, ACAT inhibitors have the potential of providing three beneficial effects in patients with hypercholesterolemia decreased cholesterol absorption, decreased hepatic VLDL synthesis, and decreased foam cell formation. Initial successes at inhibiting ACAT were dampened by the discovery of accompanying adrenal toxicity. Subsequent structural modifications have lead to the development of... 

It can be expected that all plasma lipoprotein classes, defined in one way or another, consist of a variety of subfractions, simply because plasma lipoproteins form a dynamic system. Plasma lipoprotein metabolism starts as soon as the nascent particles are secreted. Subsequent intravascular metabolism includes the actions of lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase (LCAT), and lipid transfer proteins (LTP). In addition, most lipoproteins can bind to lipoprotein receptors. This can be foUowed by uptake and irreversible intracellular degradation of the holo-particle, or by reappearance in plasma of a modified form of the lipoprotein. The modifications may be due to the transfer of cellular lipids to plasma lipoproteins or to the specific transfer of lipoprotein components to the cells. Both mechanisms may include retroendocytosis. 

The results obtained are in agreement with those reported by Lassner et al. (1995) using the LPA-AT from Limnanthes alba and demonstrate the feasibility of altering the acyl composition at specific positions of TAG in one plant species by introducing an acyltransferase with a particular selectivity from another plant species. Further modifications are required to increase the level of trieucin and erucic acid and these are the subject of current investigations. 

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