Acetyl chloroplastic

In another experiment (Table V), the free sterol content of ozonated chloroplasts from beans was found to be 32% less and the content of sterol derivatives 37% more than that of non-ozonated chloroplasts. What happens to the free sterols (FS), sterol glycosides (SG) and acetylated sterol glycosides (ASG) can be seen in Table VI, In these experiments ( ) with whole leaves of beans, FS in the ozonated leaves was 21% less, SG 32% more, and ASG 41% more than in non-ozonated leaves. 

In bacteria and in at least some plant chloroplasts,36/37 acetyl-CoA carboxylase consists of three different kinds of subunit and four different peptide chains. 

Mammalian pyruvate carboxylase has four identical subunits, and the isolated monomer will catalyze the complete reaction. By contrast, three distinct subunits can be isolated from acetyl CoA carboxylase of Escherichia coli and spinach chloroplasts a biotinyl carrier protein, biotin carboxylase, and carboxyl transferase. 

Many aroma compounds in fruits and plant materials are derived from lipid metabolism. Fatty acid biosynthesis and degradation and their connections with glycolysis, gluconeogenesis, TCA cycle, glyoxylate cycle and terpene metabolism have been described by Lynen (2) and Stumpf ( ). During fatty acid biosynthesis in the cytoplasm acetyl-CoA is transformed into malonyl-CoA. The de novo synthesis of palmitic acid by palmitoyl-ACP synthetase involves the sequential addition of C2-units by a series of reactions which have been well characterized. Palmitoyl-ACP is transformed into stearoyl-ACP and oleoyl-CoA in chloroplasts and plastides. During B-oxi-dation in mitochondria and microsomes the fatty acids are bound to CoASH. The B-oxidation pathway shows a similar reaction sequence compared to that of de novo synthesis. B-Oxidation and de novo synthesis possess differences in activation, coenzymes, enzymes and the intermediates (SM+)-3-hydroxyacyl-S-CoA (B-oxidation) and (R)-(-)-3-hydroxyacyl-ACP (de novo synthesis). The key enzyme for de novo synthesis (acetyl-CoA carboxylase) is inhibited by palmitoyl-S-CoA and plays an important role in fatty acid metabolism. 

Location. Plant fatty acid synthesis appears to be limited to chloroplasts. A chloroplast isozyme of pyruvate dehyrogenase catalyzes the conversion of pyruvate to acetyl-CoA. Pyruvate is also derived from glycerate-3-phosphate, an intermediate in the Calvin cycle, a biosynthetic pathway in which plants incorporate COz into sugar molecules. (The Calvin cycle is discussed in Chapter 13.)... 

Fatty acid synthesis in plants differs from that in animals in the following ways location (plant fatty acid synthesis occurs mainly in the chloroplasts, whereas in animals fatty acid biosynthesis occurs in the cytoplasm), metabolic control (in animals the rate-limiting step is catalyzed by acetyl-CoA carboxylase, whereas in plants, this does not appear to be the case), enzyme structure (the structures of plant acetyl-CoA carboxylase and fatty acid synthetase are more closely related to similar enzymes in E. coli than to those in animals). 

Rohmer et al. 1993). Subsequent investigations (reviewed by Lichten-thaler 1999) have shown that this pathway is widely distributed in prokaryotes, in the chloroplasts of eukaryotic algae and higher plants, and in the cytosol of members of the Chlorophyta (including the Trebouxiophyceae, Chlorophyceae, and Ulvophyceae see summary in Table 4 and note very recent clarification by Schwender et al. 2001). In contrast to acetyl-CoA, isopentenyl pyrophosphate is a dedicated product flowing only to the biosynthesis... 

Michel HP, Hunt DF, Shabanowitz J et al. Tandem mass spectrometry reveals that three photosystem II proteins of spinach chloroplasts contain N-acetyl-O-phosphothreonine at their NH2 termini. J Biol Chem 1988 263 1123-1130. 

Roughan etal 2) compared acetate, pyruvate and malonate as potential precursors and found that acetate was about three times better than pyruvate while malonate was not used at all. Consistent with this result is the report of Kuhn elal Q) who found that acetate concentration in plant tissue is in the order of mM and that acetate thiokinase is localized in the plastld. Nevertheless, alternative substrates can not be entirely excluded at this stage. Schulze-Siebert etal (4) have reported that when chloroplasts are incubated with bicarbonate, pyruvate accumulates in the chloroplast. Furthermore Williams and Randall (5) have reported that pyruvate dehydrogenase of pea chloroplasts has an activity of 6-9 pmol/h/mg chlorophyll. It is therefore conceivable that the acetyl CoA used in the first steps of fatty acid synthesis is derived from pyruvate. 

The studies of the individual enzymes of fatty acid synthesis in higher plants has shown that the two reductive steps, p-ketoacyl ACP reductase and enoyl ACP reductase have different cofactor requirements. As a result the synthesis of fatty acids depends on the availability of both NADH and NADPH. While the provision of NADPH can be attributed to the photosynthetic reactions, the source of NADH in the chloroplast is less certain. Takahama etal (8) have demonstrated that the content of NADPH in the chloroplast is influenced by illumination as expected, but there is no such fluctuation of the oxidation state of NAD/NADH. The production of NADH to be utilized in fatty acid synthesis would therefore appear to depend on dark reactions. One possibility would be by the action of pyruvate dehydrogenase, which would generate not only the NADH required for reduction in fatty acid synthesis but also the precursor acetyl CoA. 

Each plant cell manufactures its own fatty acids, since there is no lipid transport in plants. The synthesis begins with acetate which is formed from pyruvate, formed from phosphoglycerate in the Calvin cycle in leaf tissue or via degradation of sugars in seeds or fruits. De novo synthesis of fatty acids takes place in the chloroplasts of vegetative tissues or in plastids of other plant tissues. Acetate is first esterified to the -SH function of coenzyme A (CoA) via an enzyme, acetyl CoA synthetase. Coenzyme A is an adenosine derivative attached to a 4 -phosphopantetheine moiety, a chemical subunit idilch is ubiquitous in the metabolism of fatty acids. 

In the vindoline pathway (Fig. 17), the enzyme 5-adenosyl-L-methi-onine ll-methoxy-2,16-dihydro-16-hydroxytabersonine-A-methyltransferase (NMT) is localized in chloroplasts and is associated with thylakoids. Acetyl-CoA 17-0-deacetylvindoline 17-O-acetyltransferase (DAT) and desacet-oxyvindoline 17-hydroxylase (D17H) are localized in the cytosol (181). The enzymes catalyzing the two first steps from tabersonine to vindoline, TllH and 11-O-methyl transferase, are also present in the cytosol, the former being localized in the ER, the latter being soluble (241). 

Smith, 1972) and in uncharacterized particles obtained by centrifuging a 10,000 X g supernatant firaction at 144,000 x g for 1 h (Ascaho and Nicholas, 1977). In none of these studies were intact chloroplasts assayed for serine acetyltransferase. Acetyl-CoA, the substrate for the enzyme, may be generated in both chloroplasts and mitochondria (Givan and Harwood, 1976). 

In summary, synthesis of serine from CO requires the cooperation of chloroplasts and extrachloroplastic components. It is not known whether serine acetyltransferase is chloroplastic, but at least some of this enzyme activity is present in particles that are probably mitochondria. Acetyl-CoA may be generated in both chloroplasts and mitochondria. Reductive assimilation of sulfate to sulfide, and the sulfhydration of OAS occur in chloroplasts, but the quantitative significance of these reactions in chloroplasts remains to be assessed. 

Acetyl-CoA carboxylase has been purified from several plant tissues. In wheat germ, the BCCP and biotin carboxylase are associated with one fraction while the carboxyltransferase can be isolated independently (Heinstein and Stumpf, 1969). The carboxylase from chloroplasts has prokaryotic ... 

The chloroplast genes psbA and psbD encode the reaction centre polypeptides D1 and D2. The genes encode related polypeptides of 353 amino acid residues which are predicted to form five membrane-spanning regions (Fig. 2) similar to the L and M subunits of the bacterial reaction centre. However it is probable that the mature D1 polypeptide is smallo than die mature D2 polypeptide due to Ae removal of 12-16 residues at die C-terminus of DP. Both polypeptides are modified at the IV-terminus by the removal of W-formyl methionine andN-acetylation of the threonine residues. Both the D1 and D2 polypeptides may be phosphorylated on the JV-terminal threonine residue. ... 

Schwanke, unpublished Table 1). The strong decrease of fatty acid formation but not of aromatic amino acid and valine formation during chloroplast maturation indicates a diminution of the activity at the pyruvate — acetyl-CoA step. 

C/acetate (Fig. 2C) a labeling of isoprenoids were found. The fatty acid formation serves as isotopic dilution control. The mutual non-competition of pyruvate (including precursors) and acetate but the effective labeling by both substrates provides conclusive evidence that both substrates are needed to supply the introductory reaction of plastidic Mev pathway. We suggest that developing chloroplasts form the assumed acetoacetyl-CoA from acetyl-CoA and another activated decarboxylation product of pyruvate. 

The first of them is supplied by the enzyme RUBP-C/0 which catalyses the BC cycle reactions, the other one catalyses the reactions with the yet unknown enzyme x-carboxylase which leads to acetyl-CoA formation. Thus it is supposed that in chloroplasts of isoprenereleasing plants there are at least two compartments of light conversion of CO2 carbon that comes to the leaves from the air, from two different carboxilation systems or from one system but along two channels. In fig.l they are located in immediate vicinity. But the centres of carboxylation are separated, which is to be understood as a sign of the presence either of two different enzymes or two different carboxylizing centres of the same enzyme. 

In this way in the chloroplasts of isoprenereleasing plants there supposedly exist two interconnected complexes of carboxylt tion induced by light in which RUBP-C/0 supplies the pool BC with carton, while x-carboxylase supplies pool AcCoA. The primary product of cai boxylation of pool BC is F(3A and the reduced products are triozophosphates and carbohydrates. The primary product of carboxylation in AcCoA pool is unknown yet but the main product of reduction of carbon of C02 is to be considered as acetyl CoA. The coordinated functioning of these two pools depends first of all on their provision with CO2, the products of electron-transport chain of chloroplasts and on the extent of their interaction which might be realized at the level of PGA exchange. 

---