Fatty Acid Synthesis
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 C
2 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 CH
2 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 (18:1Δ
9)-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 18:1Δ
9 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 18:1Δ
9 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).