Ketoacyl-CoA synthases

Ketoacyl-CoA synthases perform the condensing step, which is the initial and rate-limiting step that determines fatty acyl specificity and results in the addition of the two-carbon moiety. These enzymes have been identified in both mice and humans. They share common evolutionarily conserved motifs in a central stretch of the amino acid sequence, although the overall sequence identity is not very high. None of the identified proteins has been crystallized or structurally determined, but structure predictions based on hydropathy plots indicate that they are polytropic proteins with five to seven transmembrane... 

Table 4. VLCFA composition of seed from wild-type Arabidopsis, and transgenic Napin-FMJSi and transgenic Napin-Jojoba Ketoacyl-CoA Synthase Arabidopsis. data from Lassner et al (1996). Values are the mol% of total fatty acids.

Lassner M. W. Lardizabal, K., and Metz J.G. (1996). A Jojoba b-ketoacyl-CoA synthase cDNA complements the Canola fatty acid Elongation Mutation in Transgenic plants. Plant Cell 8, 281-292. 

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. 

Fofana, B., Duguid, S., Cloutier, S., 2004. Qoning of fatty acid biosynthetic genes beta-ketoacyl CoA synthase, fatty acid elongase, stearoyl-ACP desaturase, and fatty acid desaturase and analysis of expression in the early developmental stages of flax (Unum usitatissimum L.) seeds. Plant Sci. 166, 1487-1496. 

A5-Eicosenoic acid P-Ketoacyl-CoA synthase and acyl-CoA desaturase Meadowfoam Overexpression Gaboon etal. (1999)... 

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. 

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. 

Acyl carrier protein (ACP) Acetyl-CoA-ACP transacetylase (AT) j3-Ketoacyl-ACP synthase (KS) Malonyl-CoA-ACP transferase (MT) )3-Ketoacyl-ACP reductase (KR) j8-Hydroxyacyl-ACP dehydratase (HD) Enoyl-ACP reductase (ER)... 

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). 

During the first reaction on fatty acid synthase, acetyl transacylase catalyzes the transfer of the acetyl group from an acetyl-CoA molecule to the SH group of a cysteinyl residue of /J-ketoacyl-ACP synthase. Malonyl-ACP is formed when malonyl transacylase transfers a malonyl group from malonyl-CoA to the SH group of the pantetheine prosthetic group of ACP (reaction 2). Then /J-keto-acyl-ACP synthase catalyzes a condensation reaction (reaction 3) in which ace-toacetyl-ACP is formed (Figure 12.14). 

The last two carbons of the fatty acid chain (i.e., those most distal from the carboxylate group) are the first introduced into the nascent chain, and acetyl-CoA can be thought of as the primer molecule of fatty acid synthesis in E. coli. The initial condensation reaction, catalyzed by P-ketoacyl-ACP synthase III (FabH), utilizes acetyl-CoA and malonyl-ACP to form the four-carbon acetoacetyl-ACP with concomitant loss of COj (Fig. 2). FabH also possesses acetyl-CoA ACP transacylase activity, and for many years it was thought that acetyl-ACP was the actual primer. However, acetyl-ACP appears to be a product of a side reaction, and the role, if any, played by this intermediate in the pathway is unknown. 

Fig. 2. Initiation of fatty acid synthesis. (1) Malonyl-CoA ACP transacylase (FabD) transfers the malonyl group from CoA to ACP and then (2) (5-ketoacyl-ACP synthase lit (FabH) catalyzes the initial irreversible condensation of malonyl-ACP with acetyl-CoA to form acetoacetyl-ACP.

Despite the presence of acetyl-CoA ACP acyltransferase activity in plant fatty acid synthase preparations, acetyl-ACP does not appear to play a major role in plant fatty acid synthesis (J. Jaworski, 1993). Instead, the first condensation takes place between acetyl-CoA and malonyl-ACP. This reaction is catalyzed by P-ketoacyl-ACP synthase III, one of three ketoacyl synthases in plant systems (Fig. 2). The acetoacetyl-ACP product then undergoes the standard reduction-dehydration-reduction sequence to produce 4 0-ACP, the initial substrate of ketoacyl-ACP synthase I. KAS I is responsible for the condensations in each elongation cycle up through that producing 16 0-ACP. The third ketoacyl synthase, KAS II, is dedicated to the final plastidial elongation, that of 16 0-ACP to 18 0-ACP. 

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