Isoprenoid Biosynthesis in Fruits and Vegetables - Postharvest Biology and Technology of Fruits, Vegetables, and Flowers - page 287

Tomatoes have two isoforms of PSY that are encoded by differentially expressed PSY
genes (Bartley and Scolnik, 1993). The accumulation of PSY1 transcripts occurs primarily
in flowers and fruits, including green fruit (Bartley and Scolnik, 1993), and is enhanced
in ripening (chromoplastic) fruit (Ronen et al., 1999). PSY2 is constitutively expressed in
all tissues, though transcripts mostly accumulate in green (chloroplastic) tissues such as
seedlings, leaves, and green fruits (Bartley and Scolnik, 1993; Fraser et al., 1999).
The partial purification of tomato PSY demonstrates its involvement in metabolite chan-
neling (Fraser et al., 2000). Although PSY is functional in a monomeric state, its native
and maximal activity is observed when it is associated with a large (at least 200 kDa)
complex containing several proteins, including IPP isomerase and GGPP synthase (Fraser
et al., 2000). The possibility exists that PSY serves to anchor the metabolic complex to the
thylakoid membrane because the hydrophilicity of the earlier enzymes (IPP isomerase and
GGPP synthase) and of the substrates (IPP and DMAPP) is greater than that of PSY and its
product (phytoene), which is directly deposited into the membrane (Fraser et al., 2000).
Phytoene synthases contain a conserved domain that includes them among the broad
category of class 1 isoprenoid biosynthesis enzymes (cd00385.1, Isoprenoid Biosyn C1,
NCBI). This superfamily includes trans -isoprenyl pyrophosphate synthases (cd00867.1,
Trans IPPS, NCBI), which use C 5 isoprene precursors to synthesize longer isoprenoids
and class I terpene cyclases (cd00868.1, Terpene cyclase C1, NCBI) that cyclize linear
isoprenoids such as geranyl-, farnesyl-, or geranylgeranyl pyrophosphate. These elongation
and cyclization reactions both form a new carbon-carbon single bond via the interaction of
an electron-deficient allylic carbocation with an electron-rich carbon-carbon double bond.
As well, all class 1 isoprenoid biosynthesis enzymes have a catalytic site that consists of
a large central cavity formed by mostly antiparallel alpha helices with two aspartate-rich
regions located on opposite walls. These residues mediate binding of prenyl phosphates via
bridging Mg 2 + ions, inducing proposed conformational changes that close the active site to
solvent, stabilizing reactive carbocation intermediates (Liang et al., 2002).
Within the trans-IPPS family, PSY belongs to a group of enzymes possessing a
conserved domain that catalyzes head-to-head (HH) (1 -1) condensations (cd00683.1,
Trans IPPS HH, NCBI). The main representative of this domain, squalene synthase (SQS,
EC 2.5.1.21), has received considerable attention because it catalyzes the first committed
step of sterol biosynthesis in mammals (Tansey and Shechter, 2000). It proceeds via a two-
step reaction in which two molecules of farnesyl pyrophosphate (C 15 ) react to form a stable
cyclopropylcarbinyl pyrophosphate intermediate (presqualene pyrophosphate), which is
then rearranged and reduced by nicotinamide adenine dinucleotide phosphate (NADPH) to
form squalene (Tansey and Shechter, 2000). PSY is similar to SQS in that it forms a cy-
clopropylcarbinyl pyrophosphate intermediate (prephytoene pyrophosphate), but it differs
in that the substrate is geranylgeranyl pyrophosphate (C 20 ) and the prephytoene pyrophos-
phate intermediate is converted to phytoene by a nonreductive rearrangement, thus NADPH
is not required. (Dogbo et al., 1988; Chamovitz et al., 1992; von Lintig et al., 1997).
There are 35 Ser, Tyr, or Thr residues dispersed throughout the PSY Trans IPPS HH
conserved domain that could be potential targets of phosphorylation as a means to regulate
PSY activity posttranslation. Many of these sites are directly adjacent to or within a few
bases of several key functional motifs. There are two DDXXD-like motifs responsible for
the coordination Mg 2 + ions located at PSY 161-DXXEDD-165 and PSY 287-DXXED-291.
The squalene synthase (SQS) Tyr171 (PSY Tyr242) residue is believed to facilitate farnesyl