Fatty acid covalent attachment

As we began this chapter, we saw that photosynthesis traditionally is equated with the process of COg fixation, that is, the net synthesis of carbohydrate from COg. Indeed, the capacity to perform net accumulation of carbohydrate from COg distinguishes the phototrophic (and autotrophic) organisms from het-erotrophs. Although animals possess enzymes capable of linking COg to organic acceptors, they cannot achieve a net accumulation of organic material by these reactions. For example, fatty acid biosynthesis is primed by covalent attachment of COg to acetyl-CoA to form malonyl-CoA (Chapter 25). Nevertheless, this fixed COg is liberated in the very next reaction, so no net COg incorporation occurs. 

The cell walls of mycobacteria contain three structures peptidoglycan, an arabinogalactan polysaccharide and long chain hydroxy fatty acids (mycolic acids) which are all covalently linked. Additional non-covalently attached lipid components found in the wall include glycolipids, various phospholipids and waxes. The lipid-rich nature of the mycobacterial wall is responsible for the characteristic acid-fastness on staining and serves as a penetration barrier to many antibiotics. Isoniazid and ethambutol have long been known as specific antimycobacterial agents but their mechanisms of action have only recently become more clearly understood. 

Levemir (Insulin detemir) Recombinant long-acting human insulin analogue produced in S. cerevisiae Engineered devoid of B30 threonine and a C14 fatty acid is covalently attached to B29 lysine Novo Nordisk 2004 (EU)... 

The three major classes of biopolymers found in eukaryotic systems are nucleic acids, proteins, and polysaccharides. The latter class is the most complex with respect to structural and stereochemical diversity. Polysaccharides indeed possess a massive information content. Furthermore, polysaccharides are commonly found in nature covalently attached (conjugated) to other biomolecules such as proteins, isoprenoids, fatty acids, and lipids.1... 

Numerous methods have been developed for attaching ligands to the surface of liposomes for reviews, see Refs. (3,6 ). For peptides, they fall into two major categories (i) covalent coupling of the peptides to preformed liposomes that contain functionalized hydrophobic anchors such as, e.g., derivatives of phos-phatidylethanolamine (PE) or (ii) incorporation of lipopeptides, obtained by conjugation of peptides to hydrophobic anchors (fatty acids, phospholipids), into liposomes either during the preparation of the vesicles or by postinsertion into preformed vesicles. In this section, we will briefly discuss these techniques and focus on the ones we have been using in our own work. 

Phosphopantetheine tethering is a posttranslational modification that takes place on the active site serine of carrier proteins - acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs), also termed thiolation (T) domains - during the biosynthesis of fatty acids (FAs) (use ACPs) (Scheme 23), polyketides (PKs) (use ACPs) (Scheme 24), and nonribosomal peptides (NRPs) (use T domain) (Scheme 25). It is only after the covalent attachment of the 20-A Ppant arm, required for facile transfer of the various building block constituents of the molecules to be formed, that the carrier proteins can interact with the other components of the different multi-modular assembly lines (fatty acid synthases (FASs), polyketide synthases (PKSs), and nonribosomal peptide synthetases (NRPSs)) on which the compounds of interest are assembled. The structural organizations of FASs, PKSs, and NRPSs are analogous and can be divided into three broad classes the types I, II, and III systems. Even though the role of the carrier proteins is the same in all systems, their mode of action differs from one system to another. In the type I systems the carrier proteins usually only interact in cis with domains to which they are physically attached, with the exception of the PPTases and external type II thioesterase (TEII) domains that act in trans. In the type II systems the carrier proteins selectively interact... 

The second family of secreted proteins that is covalently lipidated is the family of Wnt proteins. They are also involved in numerous processes like proliferation of stem cells, specification of the neural crest, and the expanding of specific cell types. The correct regulation of this pathway is important for animal development. Willert and coworkers were the first to isolate an active Wnt molecule. Mass spectroscopy studies carried out with the isolated protein revealed that cysteine 93 is palmitoylated. Mutating this amino acid to alanine led to almost complete loss of the signaling activity. Later in 2006, a second lipidation was found on a serine in Wnt3a. " In this case, the hydroxyl side chain is acylated with palmitoleic acid. This unsaturated fatty acid seems to be crucial for the progression of the protein through the secretory pathway. The attachment of two different lipid chains may therefore serve different functions. ... 

Figure 4-4. The domain organization of an integral, transmembrane protein as well as the mechanisms for interaction of proteins with membranes. The numbers illustrate the various ways by which proteins can associate with membranes I, multiple transmembrane domains formed of a-helices 2, a pore-forming structure composed of multiple transmembrane domains 3, a transmembrane protein with a single a-helical membrane-spanning domain 4, a protein bound to the membrane by insertion into the bilayer of a covalently attached fatty acid (from the inside) or 5, a glycosyl phosphatidylinositol anchor (from the outside) 6, a protein composed only of an extracellular domain and a membrane-embedded nonpolar tail 7, a peripheral membrane protein noncova-lently bound to an integral membrane protein.

Figure 8-2. Pathway for synthesis of palmitate by the fatty acid synthase (FAS) complex. Schematic representation of a single cycle adding two carbons to the growing acyl chain. Formation of the initial acetyl thioester with a cysteine residue of the enzyme preceded the first step shown. Acyl carrier protein (ACP) is a component of the FAS complex that carries the malonate covalently attached to a sulfhydryl group on its phosphopantatheine coenzyme (-SH in the scheme).

N-Myristoylation is achieved by the covalent attachment of the 14-carbon saturated myristic acid (C14 0) to the N-terminal glycine residue of various proteins with formation of an irreversible amide bond (Table l). 10 This process is cotranslational and is catalyzed by a monomeric enzyme called jV-myri s toy 11ransferase. 24 Several proteins of diverse families, including tyrosine kinases of the Src family, the alanine-rich C kinase substrate (MARKS), the HIV Nef phosphoprotein, and the a-subunit of heterotrimeric G protein, carry a myr-istoylated N-terminal glycine residue which in some cases is in close proximity to a site that can be S-acylated with a fatty acid. Functional studies of these proteins have shown an important structural role for the myristoyl chain not only in terms of enhanced membrane affinity of the proteins, but also of stabilization of their three-dimensional structure in the cytosolic form. Once exposed, the myristoyl chain promotes membrane association of the protein. 5 The myristoyl moiety however, is not sufficiently hydrophobic to anchor the protein to the membrane permanently, 25,26 and in vivo this interaction is further modulated by a variety of switches that operate through covalent or noncovalent modifications of the protein. 4,5,27 In MARKS, for example, multiple phosphorylation of a positively charged domain moves the protein back to the cytosolic compartment due to the mutated electrostatic properties of the protein, a so-called myristoyl-electrostatic switch. 28 ... 

Some membrane proteins contain one or more covalently linked lipids of several types long-chain fatty acids, isoprenoids, sterols, or glycosylated derivatives of phosphatidylmositol, GPI (Fig. 11-14). The attached lipid provides a hydrophobic anchor that inserts into the lipid bilayer and holds the protein at the membrane surface. The strength of the hydrophobic interaction between a bilayer and a single hydrocarbon chain linked to a protein is barely enough to anchor the protein securely, but many proteins have more than one attached... 

The core of the E. coli fatty acid synthase system consists of seven separate polypeptides (Table 21-1), and at least three others act at some stage of the process. The proteins act together to catalyze the formation of fatty acids from acetyl-CoA and malonyl-CoA. Throughout the process, the intermediates remain covalently attached as thioesters to one of two thiol groups of the synthase complex. One point of attachment is the —SH group of a Cys residue in one of the seven synthase proteins (j3-ketoacyl-ACP synthase) the other is the —SH group of acyl carrier protein. 

FIGURE 21-4 Acyl carrier protein (ACP). The prosthetic group is 4 -phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine contains the B vitamin pantothenic acid, also found in the coenzyme A molecule. Its —SH group is the site of entry of malonyl groups during fatty acid synthesis. 

The fatty acid chains are evidently embedded in the outer membrane as an anchor. About one-third of the lipoprotein molecules are attached covalently to the peptidoglycan through an amide linkage between the side chain amino group of the C-terminal lysine of the protein and a diaminopimelic acid residue of the peptidoglycan (Fig. 8-29). Thus, the protein replaces one of the terminal D-alanine residues of about one in ten of the murein peptides. There are 2.5 x 105 molecules of the bound form of the lipoprotein per cell spread over a surface area of peptidoglycan of 3 pm2. They appear to be associated as trimers located primarily in the periplasmic space.589... 

As the final outer stratum comeum is formed the phospholipid bilayer deteriorates and intercellular lipid layers are formed.k l These contain principally ceramides, cholesterol, and free fatty acids. Some sphingolipids are covalently attached to proteins.3... 

Other groups that can be attached covalently to enzymes include fatty acids, isoprenoid alcohols such as far-nesol, and carbohydrates. Although such modifications are widespread, our understanding of how cells use them to regulate enzymatic activities is still fragmentary. 

Numerous proteins are covalently modified by fatty acids (see Topic E2). Myristate (04 0) and palmitate (06 0) are directly attached to some proteins, while phosphatidylinositol is covalently linked to the C terminus of other proteins via a complex glycosylated structure ... 

Brennan et al. used a method to detect the reaction of acetylcholineesterase with acetylcholine [46]. The method was based on the use of a monolayer, consisting of fatty acids having Ci6 chain lengths, which were covalently attached to quartz wafers and which contained a small amount of nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE) (partitioned from water into the membrane). The enzyme substrate reaction produced a decrease in fluorescence intensity from the monolayer, and the detection system was sensitive to the changes in bulk concentration of as small as 0.1 pM, with a limit of detection of 2 pM of acetylcholine. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric methods and fluorescence microscopy. 

A role as a recognition surface in connecting proteins, similar to the role of SH2 and SH3 domains, has been postulated for PH domains. However, protein partners specifically recognizing PH domains are rare. Therefore, a role of PH domains in anchoring soluble proteins to membranes succeeded the original assumption that PH domains determine protein-protein interactions. In that case, PH domains would be like covalently attached fatty-acid residues, which also help to attach proteins to the membrane. The structural properties of PH domains support a role in bringing cytosolic proteins to the membrane. The structure of a PH domain is shown in Plate 4a. Structures of complexes of inositol phosphates with a PH domain were solved, both by X-ray crystallography and The structural feamres are shown in Plate 4b. 

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

Organisms of all biological kingdoms convert 64 into the cys-teamine derivative phosphopantetheine (65) using L-cysteine as substrate. 65 is converted to coenzyme A (66) by attachment of an adenosine moiety via a pyrophosphate linker and phosphorylation of the ribose moiety. Phosphopantetheine can be attached covalently to serine residues of acyl carrier proteins that are parts of fatty acid synthases and polyketide synthases. 

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