Agriculture Reference
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In the region between the two catalytic HKD motifs, beginning at AA 559-562 in the
seven fruit PLD alphas, is a highly conserved sequence of 25 AA, analogous to the DRY
motif in animal G-protein-coupled receptors (Wang et al., 2006). This motif in
Arabidopsis
PLD alpha 1 was shown to bind the G
α
subunit of a heterotrimeric G-protein (Zhao and
Wang, 2004). Binding of G
1 (corre-
sponding to ERF in the fruit PLDs) and resulted in the inhibition of PLD activity. Addition
of GTP restored enzymatic activity and inhibited binding of G
α
specifically involved the residues EKF in AtPLD
α
1. There is
evidence that interaction of PLD alpha and G-proteins is involved in mediation of ABA
signaling in plants (Ritchie and Gilroy, 1998), and conceivably could play a part in other
physiologically important signaling events.
With regard to the PLD alpha catalytic sites, whereas the first HKD motif appears to be
relatively isolated, the second HKD, located about 320 AAs downstream (AA 660-667 in
LePLD
α
to AtPLD
α
1; Fig. 9.12), is flanked by several motifs (including GSxN) that are important to
enzyme action, all of which together define the PLD domain. Sung et al. (1997) elegantly
demonstrated with a series of point mutations that both HKD motifs in human PLD1
are absolutely essential for catalytic activity; even highly conserved substitutions (e.g., R
for K) inactivated the enzyme. The complexity of the PLD active site becomes apparent
through several features that include substrate preference shown according to the nature of
phospholipid head groups and the degree of unsaturation in the fatty acyl chains, along with
the differing influence of various activating factors. PLD action involves the formation of a
phosphohistidine covalent enzyme intermediate (Gottlin et al., 1998) and has been proposed
to follow a ping-pong mechanism. The results of the HKD domain point mutational analyses
(Sung et al., 1997) indicated that both HKD motifs in PLD must come together and form a
single active site for enzyme function. This was confirmed in a study by Xie et al. (1998)
showing that association of the N- and C-terminal catalytic domains of rat PLD1 is required
for catalytic activity. Stuckey and Dixon (1999), on the basis of their analysis of the crystal
structure of a PLD superfamily member, proposed that the histidine residue in one HKD
motif forms the phosphoenzyme intermediate, histidine in the other HKD motif acts as
a general acid in cleavage of the phosphodiester bond, and the pair of conserved lysine
residues take part in phosphate binding.
In plant PLD alphas, flanking both sides of the second HKD domain are motifs compris-
ing the hydrophobic amino acids isoleucine, phenylalanine, and valine. These motifs may
provide the hydrophobic interaction required for fatty acid anchoring during catalysis and
thus impart the fatty acyl chain specificity of the substrates hydrolyzed. Earlier studies have
shown that medium-chain aliphatic alcohols and aldehydes such as hexanol and hexanal are
strong inhibitors of PLD action (Paliyath et al., 1999). These components are likely to exert
their inhibitory action by binding to a hydrophobic site that is essential for catalytic action.
The hydrophobic motifs flanking the second HKD motif may serve as such sites. Located
just before the first hydrophobic motif (shown in Fig. 9.12), there is another motif rich in
basic amino acids (3 R in LePLD
α
α
1, FaPLD
α
1, CmPLD
α
1, CmPLD
α
2, and CsPLD
α
1, 2
R
3) that is considered important for
the binding of anionic lipid activators of PLD including PIP
2
and PA (Dawson and Heming-
ton, 1967; Wang, 1999, 2002). These hydrophobic and positively charged motifs are highly
conserved and present in PLD alpha, beta, and gamma isoforms. A second PIP
2
-binding
site in PLD gamma, located at AA 787-791 and comprising RRVRQ (Qin et al., 1997), is
present in the modified sequences RKVNQ in LePLD
+
1 K in LePLD
α
2, and 2 R
+
1K
+
1 H in LePLD
α
α
1 (AA 740-744), and RKVNK in
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