The plastidial fatty acid synthase (FAS) is actually a complex ofmultiple dissociable components that uses malonyl-CoA generated by ACCase to build fatty acids, two carbons at a time. Malonyl-CoA:ACP transacylase first transfers the malonyl unit to acyl carrier protein (ACP), which holds acyl intermediates via a high energy thioester bond throughout the process of fatty acid synthesis. As diagrammed in Fig. 7.3, malonyl-ACP serves as the C2 donor to acceptors of various lengths in condensation reactions catalyzed by 3-ketoacyl-ACP synthases (KASes). KASIII uses acetyl-CoA as the acceptor, producing acetoacetyl-ACP; KASI acetylates 4:0-ACP through 14:0-ACP; and KASII elongates a 16:0-ACP acceptor to 3-keto-18:0-ACP. After each condensation, carbon 3 of the product has a C=O group that must be reduced to CH2 before the next condensation can occur. In the first step of this process, 3-ketoacyl-ACP reductase reduces 3-ketoacyl-ACP to 3-hydroxyacyl-ACP. 3-Hydroxyacyl-ACP dehydratase then abstracts a water molecule, producing trans-2-enoyl-ACP. Finally, enoyl-ACP reductase reduces the double bond to the requisite single bond (Fig. 7.3).
The end products of FAS are primarily 16:0- and 18:0-ACP. The latter product can be further modified by the stearoyl (18:0)-ACP desaturase, which catalyzes the formation of a cis-double bond between the C-9 and C-10 atoms of 18:0-ACP to form oleoyl (18:1Δ9)-ACP. Unlike all other fatty acid desaturases in plants, stearoyl-ACP desaturase is a soluble enzyme which has facilitated its detailed structural characterization (Lindqvist et al., 1996). The 16:0, 18:0, and 18:1Δ9 products generated in the plastid are released from ACP for export to the cytosol by the activity of two classes of acyl-ACP thioesterases, designated FatA and FatB. FatA is most active with 18:1-ACP, whereas FatB is most active with 16:0-ACP (Salas and Ohlrogge, 2002). By the combined activities of FatA and FatB, 16:0, 18:0, and 18:1Δ9 are made available for further modification and ultimately for storage in TAG molecules by ER-localized enzymes. The stearoyl-ACP desaturase and acyl-ACP thioesterases will be discussed further because they represent major biotechnological targets for alteration of the saturated fatty acid content of seed oils. In addition, structurally variant forms of these enzymes have arisen in seeds of certain plants and are involved in the synthesis of unusual fatty acids, many of which have potential economic value (Voelker and Kinney, 2001).
Of the FAS components, KASIII has been considered a likely gatekeeper, since the Escherichia coli homologue is inhibited by acyl-ACPs, the products of FAS (Heath and Rock, 1996). Similar feedback inhibition has been observed in vitro for the KASIII of Cuphea lanceolata, a plant that produces an unusual proportion of caprylic acid (8:0) (Brück et al., 1996). However, Dehesh et al. report that overexpression of spinach KASIII in rapeseed actually reduced both FAS activity and oil content of seeds (Dehesh et al., 2001). Based on elevated acetoacetyl-ACP in leaves of tobacco transformed with the same gene, as well as increased 16:0 accumulation in both organs, they propose that reduced supplies of malonyl- ACP to KASI and KASII are responsible. It should also be noted that, in vitro, Cuphea KASes can decarboxylate malonyl-ACP under conditions promoting accumulation of 3-ketoacyl-ACP (Winter et al., 1997).
Reduced expression of several individual components of FAS decreases overall fatty acid synthesis in plants. Interestingly, when rapeseed 3-ketoacyl-ACP reductase mRNA and protein were decreased using antisense methods, enoyl- ACP reductase mRNA and protein were also downregulated (Slabas et al., 2002). This is consistent with evidence that ratios of FAS components remain unchanged during rapeseed development (O'Hara et al., 2002). Expression of FAS genes during seed maturation likewise appears coordinated with that of genes for ACCase and other enzymes related to oil production, suggesting the participation of global transcription factors comparable to the FasR factor that upregulates fatty acid synthesis in E. coli (Cronan and Subrahmanyam, 1998; Lee et al., 2002; Ruuska et al., 2002; Slabas et al., 2002).
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