3-ketoacyl-CoA synthase

Elol, Elo2, and Elo3 are 3-ketoacyl-CoA synthases embedded in the ER [8] and involved in catalyzing the four-step cycle that successively elongates precursor acyl CoA by two methylene groups. Ejsing et al. [19] performed a comparative... 

Guo, Y.M., Mietkiewska, E., Francis, T., Katavic, V., Brost, J.M., Gibhn, M., Barton, D.L. and Taylor D.C. 2009. Increase in nervonic acid content in transformed yeast and transgenic plants by introduction of a Lunaria annua L. 3-ketoacyl-CoA synthase (KCS) gene. Plant Mol. Biol. 69 565-575. 

The analysis of reaction products by thin layer chromatography suggested that this molecule could act on the condensation reaction. This was demonstrated directly by measuring only the 3-ketoacyl-CoA synthase in presence of increasing concentrations of dioxolane-CoA the level of inhibition was similar to that observed on the overall elongation process. 

Blacklock, B.J., Jaworski, J.G., 2002. Studies into factors contributing to substrate specificity of membrane-botmd 3-ketoacyl-CoA synthases. Etrr. J. Biochem. 269, 4789-4798. 

Han, J., Liihs, W., Sonntag, K., Zahringer, U., Borchardt, D.S., Wolter, F.R, Heinz, E., Frentzen, M., 2001. Functional characterization of 3-ketoacyl-CoA synthase genes from Brassica napus L. Plant Mol. Biol. 46,119-2 9. 

FIGURE 25.7 The pathway of palmhate synthesis from acetyl-CoA and malonyl-CoA. Acetyl and malonyl building blocks are introduced as acyl carrier protein conjugates. Decarboxylation drives the /3-ketoacyl-ACP synthase and results in the addition of two-carbon units to the growing chain. Concentrations of free fatty acids are extremely low in most cells, and newly synthesized fatty acids exist primarily as acyl-CoA esters. 

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). 

Fig. 2.3 Biosynthesis pathway of A P(3HB) B P(3HB-co-3HV) C P(3HB-co-3HHx) via fatty acid /S-oxidation and D P(3HB-co-3HHx) via fatty acid de novo synthesis. PhaA, f -ketothiolase PhaB, NADPH dependent acetoacetyl-CoA reductase PhaC, PHA synthase PhaG, 3-hydroxyl-ACP-CoA transferase PhaJ, (J )-enoyl-CoA hydratase FabG, 3-ketoacyl-CoA reductase (Sudesh et al. 2000)...

The second pathway involves the production of PHAs via the fatty acid degradation pathway. In this case, the resulting monomers in the polymer chain were similar in structure to the carbon source or shortened by 2, 4 or 6 carbon atoms [29]. In this pathway the fatty acids are first converted to the corresponding acyl-CoA which are then oxidised by the /1-oxidation pathway via enoyl-CoA, (5)-3-hydroxyacyl-CoA and 3-ketoacyl-CoA precursors. Finally, enzymes like the enoyl-CoA hydratase, hydroxyacyl-CoA epimerase, and /3-ketoacyl-CoA reductase connect the /1-oxidation pathway with the medium-chain length PHA biosynthesis through the PHA synthase [30]. 

It has also been demonstrated that 3-ketoacyl-ACP reductase (FabG) can accept not only acyl-ACP but also acyl-CoA as a substrate and is capable of supplying mcl-(R)-3HA-CoA from fatty acid p-oxidation in Escherichia co/i. FabG, which is known as a homologue of PhaB, has been reported to serve as a monomer supplier for PHA biosynthesis in recombinant E. colt. 3-Ketoacyl-AGP synthase III (FabH) is also a constituent of fatty... 

Fig. 2 Metabolic routes for mcl-PHA biosynthesis. Pseudomonas putida GPol synthesizes PHA through P-oxidation and P. putida KT2440 synthesizes PHA through fatty add de novo synthesis. Special PHA consisting of 4-hydroxyalkanoate, 5- hydroxyalkanoate, or 6-hydroxyalkanoate can be produced by various bacteria when suitable precursors are supplied. 1 acyl-CoA synthetase, 2 acyl-CoA dehydrogenase, 3 enoyl-CoA hydratase, 4 NAD-dependent (5)-3-hydroxyacyl-CoA dehydrogenase, 5 3-ketoacyl-CoA thiolase, 6 (ItFspecific enoyl-CoA hydratase, 7 NADPH-dependent 3-ketoacyl-CoA reducatase, 8 3-hydroxyacyl-CoA epimerase, 9 mcl-PHA polymerase, 10 acetyl-CoA carboxylase, 11 malonyl-CoA-acyl carrier protein (ACP) tiansacylase, 12 3-keto-ACP synthase, 13 3-keto-ACP reductase, 14 3-hydroxyacyl-ACP dehydratase, 15 enoyl-ACP reductase, 16 acyl-ACP thiolase, 17 (l )-3-hydroxyacyl-ACP-CoA transacylase, 18 mcl-PHA polymerase...

The acetyl group on the cysteine residue condenses with the malonyl group on ACP as the CO2 originally added by acetyl CoA carboxylase is released. The result is a four-carbon unit attached to the ACP domain. The loss of flee energy from the decarboxylation drives the reaction catalyzed by 3-Ketoacyl-ACP synthase ... 

In subsequent steps of fatty acid synthesis, a fatty acyl group that is linked by a thioester bond to the active site of 3-ketoacyl-ACP synthase condenses with malonyl-ACP. One molecule of CO2 is liberated (Browse and Somerville, 1991 Conn and Stumpt, 1972, Lehninger, 1982) (Fig. 2.4). Although acetyl-ACP has been considered to condense initially with malonyl-ACP, recent work indicates that the product of the first condensation, butyryl-ACP, is formed by the condensation of acetyl-CoA and malonyl-ACP and that acetyl-ACP is a minor participant in fatty acid biosynthesis (Jaworski et al., 1993). 

Three forms of 3-ketoacyl-ACP synthase have been discovered in plants. These forms may be distinguished by their substrate specificity they are homodimers with molecular weights of 43,000 to 45,000 per subunit. One, KAS III, appears to be responsible for the first condensation of acetyl-CoA and malonyl-ACP (Browse and Somerville, 1991 Ohlrogge et al., 1993). The activity of this enzyme in plants seems to bypass the need for acetyl-ACP, although that molecule is formed and accumulated in some plants (Ohlrogge et al., 1993). KAS I or 3-ketoacyl-ACP synthase elongates the acyl chain to palmitoyl-ACP, whereas KAS II converts palmitoyl-ACP to stearoyl-ACP (Ohlrogge et al., 1993). 

In fatty acid synthesis or degradation (Fig. 4), once the substrate is in the form of alkanoyl-CoA, a two-step process involving a 3-ketoacyl-CoA reductase and a PHA synthase occurs. The reduction step requires NADPH. A suitable supply of intracellular substrate appears to be the key factor controlling the rate of biosynthesis. If P. oleovorans is fed octanoate, it accumulates much more PHA than if hexanoate or dodecanoate are added. Nevertheless, the S5mthase is relatively nonspecific. P. oelovorans can accumulate polymers in which the side chain of the monomers contain double bonds, branches, cyclic structures, or even halogen atoms if the appropriate substrate is supplied. 

Figure 7.7 Common metabolic pathways that are involved in the biosynthesis of PHA in microorganisms. FabC malonyl-CoA acyl carrier protein (ACP) transcylase, FabD malonyl-CoA-ACP transacylase, FabG 3-ketoacyl-CoA reductase, PhaA P-ketothiolase, PhaB NAOH-dependent acetoacetyl-CoA reductase, PhaC polyhydroxyalkanoates synthase, PhaG 3-hydroxyacyl-ACP GoA transferase, PhaJ (R)-enoyl-GoA hydratase and TCA tricarboxylic acid...

Although it was known that the intermediates of the yS-oxidation cycle are chaimelled towards PHA biosynthesis, only recently the precursor sources were identified. In A. caviae, the y3-oxidation intermediate, trans-2-tnoy -CoA is converted to (R)-3-hydroxyacyl-CoA via (R)-specific hydration catalysed by an (R)-specific enoyl-CoA hydratase [125, 126]. Subsequently, Tsuge and co-workers [127] reported the identification of similar enoyl-CoA hydratases in Pseudomonas aeruginosa. In the latter case, two different enoyl-CoA hydratases with different substrate specificities channelled both SCL and MCL enoyl-CoA towards PHA biosynthesis. In recombinant . coli it was further shown that 3-ketoacyl-CoA intermediates in the )8-oxidation cycle can also be channelled towards PHA biosynthesis by a nicotinamide adenine dinucleotide phosphate dependent (NADPH-dependent) 3-ketoacyl-ACP reductase [128]. A similar pathway was also identified in P. aeruginosa [129]. In addition, it was also reported that the acetoacetyl-CoA reductase (PhaB) of R. eutropha can also carry out the conversion of 3-ketoacyl-CoA intermediates in Pathway II to the corresponding (R)-3-hydroxyacyl- CoA in E. coli [130]. The results clearly indicate that several channelling pathways are available to supply substrates from the y3-oxidation cycle to the PHA synthase. This explains why it was not possible to obtain mutants that completely lack PHA accumulation ability, unless the mutation occurred in the PHA synthase gene [131]. 

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