De novo fatty acid biosynthesis

Previously, we determined that the only known mode of action that is selective for grasses/monocots is acetyl CoA carboxylase inhibition.6 This enzyme is the first of two enzymes involved in de novo fatty acid biosynthesis. This mode of action prevents the synthesis of many essential wax compounds. In order to screen our compounds for this mode of action, we used resistant oat (Avena sp.) seeds. The emodin analogues caused dose-dependent bleaching (Fig. 1.7) and a severe decrease in germination for both resistant and nonresistant grasses. 

We decided to focus our attention on de novo fatty acid biosynthesis based upon our observations and those reported in the literature (12). To determine whether fatty acid biosynthesis was inhibited, we determined the effect of the herbicides on incorporation into palmitic (16 0) acid, the first product of fatty acid biosynthesis. Maize leaf discs were incubated for 40 min in 14C -acetate, after which fatty acids were extracted. As shown in Table II, both tralkoxydim and haloxyfop significantly reduced the amount of acetate incorporated into palmitic acid. Thus, these compounds affected a step early in lipid biosynthesis. 

De novo fatty acid biosynthesis from cytosolic citrate... 

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 ... 

The answer is a. (Murray, pp 123-148. Scriver, pp 2367-2424. Sack, pp 159—175. Wilson, pp 287-317.) Acetyl CoA carboxylase deficiency drastically alters the ability of the patient to synthesize fatty acids. The fact that the infant was born at all is due to the body s ability to utilize fatty acids provided to it. However, all processes dependent upon de novo fatty acid biosynthesis are affected. The lungs, in particular, require surfactant, a lipoprotein substance secreted by alveolar type 11 cells, to function prop-... 

Greseth, M. D., Traktman, P. (2014). De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection. PLoSPathog, 10(3), el004021. doi 10.1371/joumal.ppat.l004021... 

Ohlrogge, J. B., J. G. Jaworski, and D. Post-Beittenmiller, De novo fatty acid biosynthesis, in Lipid Metabolism in Plants (T. S. Moore, Jr., ed.) 3-32, CRC Press, Boca Raton, FL, 1993. 

Biosynthesis of MCL-PHAs involves three different pathways namely (1) de novo fatty acid biosynthesis, (2) P-oxidation pathway and (3) chain elongation pathway, which is elucidated in Fig. 8.4. 

De novo fatty acid biosynthesis StmcturaUy unrelated carbon sources such as glucose generate (R)-3-hydroxyacyl-CoA precursors via this metabolic route. (R)-3-hydroxyacyl-ACP is converted to (/ )-3-hydroxyacyl CoA in a reaction, which is catalysed by the enzyme (R)-3-hydroxyacyl-CoA-ACP transferase. This enzyme is encoded by the phaG gene (Sudesh et al., 2000). 

As expected, since cd obtains the majority of its fatty acid from the supplement, cd incorporated a greater proportion of exogenous DHS into its lipids than did wild type. In addition, DHS treatment inhibited the formation of the longer-chain polyunsaturates. When added at the time of inoculation, DHS also promoted growth and de novo fatty acid biosynthesis by cd to near wild type. We are currently investigating the mechanism of this effect. 

In plants, de novo fatty acid biosynthesis occurs exclusively in the stroma of plastids, whereas, with the exception of plastidial desaturation, modification of fatty acid residues including further desaturation and triacylglrycerol (TAG) assembly are localized in the cytosol/endoplasmic reticulum (ER). The primary fatty acids formed in the plastid (palmitic, stearic, and oleic acid) are used in the plastidic prokaryotic pathway for membrane lipid synthesis or diverted to the cytoplasmic eukaryotic pathway for the synthesis of membrane lipids or storage TAGs (1). Movement of glycerolipids is believed to occur in the reverse direction between the cytosol/ER and the plastids in the highly regulated manner (2). 

Fig.4 Inhibitory activity of the esterified and free acid-form of diclofop and haloxyfop on de novo fatty acid biosynthesis of oat chloroplasts.

All the different effects of cyclohexanedione or diphenoxypropionic acid-type herbicides can be explained by their inhibition of de novo fatty acid biosynthesis at the level of the acetyl-CoA carboxylase as main target since neither lipid nor biomembrane formation or growth are possible when fatty-acid biosynthesis is blocked. 

Fig. 9 Scheme of the inhibition by xenobiotics of different steps of de novo fatty-acid biosynthesis. 

Today several xenobiotics, either natural antibiotics (cerulenin, thiolactomycin) or active herbicides are known to interfere with acetyl-CoA formation, de novo fatty acid biosynthesis, desaturation of fatty acids, biosynthesis of long chain fatty acids and glycerolipid formation. This is summarized in Fig. 9. Certainly other new herbicides which interfere with the same or other particular parts of plant lipid biosynthesis will be developed in the future. From the study of the mode of action of these inhibitors in sensitive and tolerant species, one will not only be able to efficiently control weeds in crop plants and guarantee a better food production but also obtain a new and better understanding of the function of plant lipids and the regulation of plant lipid biosynthesis. 

The de novo fatty acid biosynthesis of isolated oat chloroplasts as well as its inhibition by cycloxydim was dependent on the age of the chloroplasts. The youngest oat chloroplasts isolated from the lower part of the primary leaf blades had the highest capacity for fatty acid synthesis which was inhibited by IpM cycloxydim to a larger degree than in older chloroplasts. In those chloroplasts, isolated from the middle and upper part of the... 

CHEMICAL REGULATION OF ACETYL-COA FORMATION AND DE NOVO FATTY ACID BIOSYNTHESIS IN PLANTS... 

There is still discussion which enzyme, ACS or pPDHC, provides the major acetyl-CoA pool for de novo fatty acid biosynthesis, and this seems to be dependent on plant species, development of plants and plastids and on the plastidic levels of pyruvate and acetate [4,6]. In order to obtain new information on the contribution of both enzymes to de novo fatty acid biosynthesis we looked for enzyme activities in developing chloroplasts and for specific inhibitors of the ACS and the pPDHC. The tests were performed in isolated chloroplasts and etioplasts which are capable of incorporating exogenously applied [ C]-acetate as well as [2- C]-pyruvate Into the total fatty acid fraction [6,7]. A specific, potent Inhibitor of the plant ACS was found to be ethyl-adenylate (ethyl-AMP) (Figure 1) which mimics the tightly enzyme-bound ACS-intermedlate acetyl-adenylate [7]. acetylmethylphosphinate (Figure 1) is a known inhibitor of mitochondrial PDHC [8] which also blocks the plastidic pPDHC as is shown here. We also demonstrate that etioplasts prefer pyruvate and chloroplasts acetate for fatty acid biosynthesis. 

With ethyl-AMP, we found a specific inhibitor of plant ACS which is competitive with respect to ATP (Kj-value 42 nM) and non-competitive with respect to acetate [6]. Ethyl-AMP inhibits plant ACS preparations (l5o-value 0.4 pM) but also de novo fatty acid biosynthesis in intact plastids starting from acetate (l5Q-value 10 pM in isolated radish etioplasts). The incorporation of labelled pyruvate in etioplasts was not affected by ethyl-AMP. 

With ethyl-AMP and AMPI specific inhibitors of the two independent routes of acetyl-CoA formation in plastids are available. Several specific xenobiotics block efficiently de novo fatty acid biosynthesis at different steps and enzyme levels (Figure 3). Graminicides such as diclofop, sethoxydim or cycloxydim are specific inhibitors of acetyl-CoA carboxylase (ACCase) of grasses [10], the antibiotics cerulenin and thiolactomycin are inhibitors which affect certain of the li-ketoacyl-ACP synthases (KAS I, II and III). With these xenobiotics one can control the metabolite flow through the fatty acid biosynthesis pathway and obtain a better understanding of the regulation of the plants de novo fatty acid biosynthesis and the enzymes involved. 

Figure 3 Scheme of de novo fatty acid biosynthesis pathway from acetate and pyruvate with indication of the site of action of several inhibitors. 

Topfer R. Martini N. Molecular cloning of cDNAs or genes encoding proteins involved in de novo fatty acid biosynthesis in plants. J Plant Physiol 1994 143 416 -425. 

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