Agriculture Reference
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by other phospholipids. Presumably, Ca
2
+
binding in the catalytic domain of PLD alphas
could play a part in activation, but this has not been determined experimentally.
Among the seven fruit PLD alphas aligned in Fig. 9.12, a small region of structural
diversity at AAs 23-25 (26-29 in FaPLD
1) is observed at the beginning of the second beta-
sheet sequence (note that this is the start of the first Ca
2
+
-binding loop in the alignment based
on PLC delta 1 and cPLA
2
depicted in Zheng et al., 2000). Typically this region is occupied
by three glycine and/or serine residues, as is the case in CmPLD
α
α
1, CsPLD
α
1, CmPLD
α
2,
and FaPLD
3 differ by the presence of asparagine followed by
the hydrophobic amino acid phenylalanine. In marked contrast, C2 of LePLD
α
1. LePLD
α
2 and LePLD
α
1 contains
three acidic glutamate residues (preceding the more typical GGH). Recent investigations
have shown that a tomato PLD alpha 1 C2-GFP chimeric protein expressed in
Eschericiacoli
show calcium-dependent association with microsomal membrane vesicles below 1
α
μ
Mlevel
of calcium confirming the high affinity of the PLD alpha 1 C2 domains to calcium (Tiwari and
Paliyath, 2007). There is some evidence that the N-terminal 30-35 AAs compose a leader
sequence that is clipped from the mature enzyme in vivo (Wang, 2000), so possibly the
diverse region within this leader sequence serves a targeting function, directing localization
of different PLD alpha isozymes.
Another characteristic feature of PLD alphas that is likely to confer significant secondary
structure is two highly conserved motifs including a relatively large number of positively
charged, basic amino acids (arginine; R and lysine; K). The first motif lies within the C2
domain (Qin et al., 1997) and among the seven fruit PLD alphas (Fig. 9.12) includes 8-9
residues beginning at AA 56-59. The sequence is KARVGRTR in LePLD
α
1, LePLD
α
2,
α
α
α
α
LePLD
3, CmPLD
1, and CsPLD
1, KARVGRTRR in CmPLD
2, and RARVGRTR
in FaPLD
1. The second, 21-AA motif ranges from AA 239-242 to AA 259-262 and
includes 3 R
α
+
3 K in CmPLD
α
1 and CsPLD
α
1,3R
+
4 K in LePLD
α
1, LePLD
α
2,
and FaPLD
3. By virtue of their
consistent presence and conserved nature, these motifs are likely to be of great signifi-
cance to the conformation of PLD alpha. The abundance of positively charged residues
may serve as an anchoring domain that binds to anionic lipid domains in the membrane
composed of polyphosphoinositides (PIP, PIP
2
, PIP
3
). We propose that calcium binding
to the C2 domain could expose these positively charged domains, facilitating binding to
polyphosphoinositide-enriched, anionic membrane domains that are generated in response
to primary stimuli. Further experimentation will be required to test this hypothesis.
Beyond the second positively charged, arginine- and lysine-rich motif, in the catalytic
portion of PLD alpha, lie the two active site domains. Each has the strictly conserved
HxKxxxxD motif, but the second also includes another conserved group of AAs, GSxN, such
that the entire motif at the second active site is HxKxxxxDxxxxxxGSxN (Fig. 9.12). Also
referred to as the phospholipase D domain (Swiss Institute of Bioinformatics), this motif is
common to a broad array of phosphodiesterases that make up the PLD superfamily (Ponting
and Kerr, 1996; Stuckey and Dixon, 1999; Wang, 2000). In the seven PLD alphas shown in
Fig. 9.12, the first HxKxxxxD motif occurs over a range varying from AA 330-337 to AA
333-340, and among these seven sequences it differs only by the conserved substitution of
one isoleucine for one valine in LePLD
α
1, 3 R
+
5 K in CmPLD
α
2, and 5 R
+
3 K in LePLD
α
α
1 and LePLD
α
3. Ten AAs downstream from this
active site (AA 349-351 in LePLD
α
1), there is another positively charged motif consisting
α
α
α
α
α
of KKR in LePLD
1, CsPLD
1, and CmPLD
2, RRR in CmPLD
1, LePLD
2, and
LePLD
α
3, and SRR in FaPLD
α
1.
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