E. coli ACP

Because E. coll ACP is readily available and is the most active of all ACPs tested, it usually has been employed in experiments with plant fatty acid synthetases. For de novo synthesis, E. coli ACP does not appear to modify the normal products of synthesis by plant synthetases. 

Stearoly-ACP ( . coli) is rapidly desaturated to oleoyl-ACP by plant stearoyl-ACP desaturases. However, antibodies to E. coli ACP cross-react very poorly with spinach ACP (Ohlrogge et al., 1979). Conversely antibodies to spinach ACP cross-react poorly with E. coli ACP. 

Table 1 shows that the fusion protein was active in the standard assay for ACP, but that reactivity was not comparable to E. coli ACP at lower concentrations (up to 4 lg added to 50 X reaction 8.5 XM). We have calculated that the Km app for the fusion ACP was 0.5 mM and the equivalent figure for E. coli ACP was 50 jiM. This 10-fold increase in Km value suggests that the ACP portion of the fusion requires higher concentration to be recognized by the acyl ACP synthetase. This effect is probably caused by the unrecognized portion (Ca 75% by mass) taken up by protein A. During the linear phase of reactivity (i.e., between 1.6 to 4.0 lg 50 it appears that the fusion... 

As shown in Table 1, of individual six enzymes from E. coli ACP acetyltransferase and 3-ketoacyl-ACP synthase were inhibited more than 50 % at 3-5 pM TLM, although the other enzymes retained full activities at this concentration. TLM reversibly inhibited fatty acid synthase. The inhibition of acetyltransferase was competitive for ACP, but uncompetitive for acetyl-CoA. The inhibition of 3-ketoacyl-ACP synthase was also competitive for malonyl-ACP but noncompetitive for acetyl-ACP (Fig. 1). 

A PEG precipitated homogenate of avocado was prepared and subjected to anion exchange on a Pharmacia Mono Q column. 0.5ml fractions were collected and assayed for binding of [1-f C] from [1-f"fC] acetyl CoA in the presence and absence of E.coli ACP. The first peak was absolutely dependent on the presence of ACP for incorporation of [1- C], confirming this... 

FIGURE 25.13 Double bonds are introduced into the growing fatty acid chain in E. coli by specific dehydrases. Palmitoleoyl-ACP is synthesized by a sequence of reactions involving four rounds of chain elongation, followed by double bond insertion by /3-hydroxydecanoyl thioester dehydrase and three additional elongation steps. Another elongation cycle produces cA-vaccenic acid. 

It is worth mentioning that metabolic engineering of E. coli recently provided recombinant strains which synthesized PHAMCL from gluconate. For this, beside phaC2Po or phaClPa> the thioesterase I from E. coli (TesA) [128] or the acyl-ACP thioesterase from Umbellularia californica [129], respectively, were expressed in E. coli. However, the amounts of PHAMCL accumulated in the cells were rather low, and this artificial pathway was not very efficient. 

Thiolactomycin (16) is another natural product that reversibly inhibits E. coli FabF, FabB, and FabH with respective ICso s of 6, 25 and 110 (iM. Unlike cerulenin, it binds the malonyl-ACP site of the enzyme [27]. Despite modest double-digit MICs on . coli, S. aureus, Serratia marces-cens, and Mycobacterium tuberculosis, 16 has generated quite some interest due to its good in vivo protection against an oral or intramuscular S. marcescens urinary tract infection model where it displayed rapid tissue distribution [28]. Despite several medicinal chemistry efforts, thiolactomycin has proven difficult to optimize due to some strict functional group requirements for its SAR [29]. 

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. 

In E. coli and some plants, the seven active sites for fatty acid synthesis (six enzymes and ACP) reside in seven separate polypeptides (Fig. 21-7, top). In these complexes, each enzyme is positioned with its active site near that of the preceding and succeeding enzymes of the sequence. The flexible pantetheine arm of ACP can reach all the active sites, and it carries the growing fatty acyl chain from one site to the next intermediates are not released from the enzyme complex until it has formed the finished product. As we have seen in earlier chapters, this channeling of intermediates from one active site to the next increases the efficiency of the overall process. 

The fatty acid synthases of yeast and of vertebrates are also multienzyme complexes, and their integration is even more complete than in E. coli and plants. In yeast, the seven distinct active sites reside in two large, multifunctional polypeptides, with three activities on the a subunit and four on the /3 subunit. In vertebrates, a single large polypeptide (Afr 240,000) contains all seven enzymatic activities as well as a hydrolytic activity that cleaves the finished fatty acid from the ACP-like part of the enzyme complex. The vertebrate enzyme functions as a dimer (Afr 480,000) in which the two identical subunits lie head-to-tail. The subunits appear to function independently. When all the active sites in one... 

A closely related E. coli protein is a 79-kDa multifunctional enzyme that catalyzes four different reactions of fatty acid oxidation (Chapter 17). The amino-terminal region contains the enoyl hydratase activity.32 A quite different enzyme catalyzes dehydration of thioesters of (3-hydroxyacids such as 3-hydroxydecanoyl-acyl carrier protein (see Eq. 21-2) to both form and isomerize enoyl-ACP derivatives during synthesis of unsaturated fatty acids by E. coli. Again, a glutamate side chain is the catalytic base but an imidazole group of histidine has also been implicated.33 This enzyme is inhibited irreversibly by the N-acetylcysteamine thioester of 3-decynoic acids (Eq. 13-8). This was one of the first enzyme-activated inhibitors to be studied.34... 

Both bacteria and plants have separate enzymes that catalyze the individual steps in the biosynthetic sequence (Fig. 17-12). The fatty acyl group grows while attached to the small acyl carrier protein (ACP).54 58 Control of the process is provided, in part, by the existence of isoenzyme forms. For example, in E. coli there are three different P-oxoacyl-ACP synthases. They carry out the transfer of any acyl primer from ACP to the enzyme, decarboxylate malonyl-ACP, and carry out the Claisen condensation (steps b, e, and/in Eq. 17-12)58a e One of the isoenzymes is specialized for the initial elongation of acetyl-ACP and also provides feedback regulation.58c The other two function specifically in synthesis of unsaturated fatty acids. 

Fatty acid synthesis begins when the substrates, acetyl-CoA and malonyl-CoA, are transferred onto the protein by malonyl-CoA acetyl-CoA-ACP transacylase (MAT, steps 1 and 2 in fig. 18.12a). The numbers in parentheses below the abbreviation of the enzyme in this figure refer to the reactions shown in fig. 18.12. (Whereas E. coli has separate enzymes that catalyze the transfer of acetyl- and malonyl-CoA to ACP, both reactions are catalyzed by the same enzymatic activity (MAT) on the animal fatty acid synthase.) Subsequently, /3-ketobutyryl-ACP and CO2 are formed in a condensation reaction catalyzed by /3-ketoacyl-ACP synthase (KS, step 3 in fig. 18.12a). 

Anaerobic pathway for biosynthesis of monounsaturated fatty acids in E. coli. Synthesis of monounsaturated fatty acids follows the pathway described previously for saturated fatty acids until the intermediate j8-hydroxydecanoyl-ACP is reached. At this point an apparent competition arises between the enzymes involved in saturated and unsaturated fatty acid synthesis. 

As the name anaerobic implies, the double bond of the fatty acid is inserted in the absence of oxygen. Biosynthesis of monounsaturated fatty acids follows the pathway described previously for saturated fatty acids until the intermediate /3-hydroxydecanoyl-ACP is reached (fig. 18.15). At this point, a new enzyme, /3-hydroxydecanoyl-ACP dehydrase, becomes involved. This dehydrase can form the a-j8 trans double bond, and saturated fatty acid synthesis can occur as previously discussed. In addition, this dehydrase is capable of isomerization of the double bond to a cis /3-y double bond as shown in figure 18.15. The /3-y unsaturated fatty acyl-ACP is subsequently elongated by the normal enzymes of fatty acid synthesis to yield pal-mitoleoyl-ACP (16 1A9). The conversion of this compound to the major unsaturated fatty acid of E. coli, cA-vacccnic acid (18 1A11), requires a condensing enzyme that we have not previously discussed, /3-ketoacyl-ACP synthase II, which shows a preference for palmitoleoyl-ACP as a substrate. The subsequent conversion to vaccenyl-ACP is cata-... 

Fig. 2. Metabolic pathways for PHA biosyntheis in fad mutant E. coli strains used in this study. Enoyl-CoA hydratase, epimerase, and 3-ketoacyl-CoA or ACP reductase have been suggested to supply PHA precursors from inhibited b-oxidation pathway. The crosses indicate inactivation of corresponding enzymes. The question mark represents uncharacterized enzyme. Enzymes involved in the metabolic pathways shown have been described previously FabG (21,32), YfcX (24,33), MaoC (34), PhaA (36), and PhaB (36).

Each of the enzymatic activities located in a single polypeptide chain of the mammalian fatty acid synthetase exists as a distinct protein in E. coli. The acyl-carrier protein (ACP) of E. coli has an Mr = 8,847 and contains 4-phosphopantotheine. The dehydratase has a molecular weight of 28,000 and catalyzes either trans 2-3 or cis 3-4 dehydration of the hydroxy acid intermediates in the biosynthesis of palmitic acid. When the chain length of the hydroxy fatty acid is C[ the synthesis of palmitoleic acid is achieved as follows ... 

The last step in the fatty acid biosynthetic pathway is catalyzed by enoyl-acyl carrier protein (ACP) reductase, which is responsible for reduction of the double bond in the enoyl-ACP derivative (Heath and Rock, 1995 Payne et al., 2002). While fabl genes encode enoyl-ACP reductases (FabI enzymes) in S. aureus and E. coli, an alternative enoyl-ACP reductase, FabK, replaces the function of Fabl in a number of bacterial species such as Streptococcus pneumoniae (Heath and Rock, 2000). More interestingly, a number of bacterial species (such as Enterococcus faecalis and Pseudomonas aeruginosa) possess both the Fabl and FabK enzymes (Heath and Rock, 2000). To discover Fabl-specific antibacterial inhibitors, Payne and colleagues at GlaxoSmithKline (GSK) developed assays for various versions of enoyl-ACP reductases (Payne et al., 2002 Seefeld et al., 2003) based on the following reaction scheme ... 

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