Agriculture Reference
In-Depth Information
for the different isozymes in a single species. It should be noted that stacking of sequences
within the phylogram does not necessarily reflect the closest structural similarity;
FaPLD
α
1
α
is most similar to
PLD
1
from castor bean (
Ricinus communis
), but is also quite similar
to
CmPLD
1
. A PLD alpha from grape berry (
Vitis vinifera
;
ABC59316) aligned most closely with castor bean
PLD
α
1
,
CsPLD
α
1
, and
LePLD
α
α
1
, peanut (
Arachis hypogaea
)
PLD
α
2
(BAE79735), and
CmPLD
α
1
. Aside from each other,
LePLD
α
2
and
LePLD
α
3
are
most similar to peanut
PLD
α
2
and castor bean
PLD
α
1
.
9.6.1 Chemical and physical parameters of fruit PLD alphas
From
the
predicted
amino
acid
sequences
for
PLD
alphas
encoded
by
genes
in
α
α
α
α
tomato (
LePLD
1
,
LePLD
2
, and
LePLD
3
), strawberry (
FaPLD
1
), honeydew melon
(
CmPLD
1
), several structural and functional
properties of these proteins were obtained using programs available on the world wide web.
Table 9.3 presents a comparison of the seven PLD alphas with respect to the number of
amino acids, molecular mass, sums of aspartate plus glutamate and arginine plus lysine
residues, acidity, isoelectric point (pI), and grand average hydropathicity. At a first glance,
the remarkable similarity of these highly conserved protein sequences is clearly evident.
The total amino acids and molecular mass vary by only 0.5 and 1.4%, respectively. There
are, however, some interesting differences in acidity and pI, which are determined by the
relative numbers of charged acidic and basic amino acids (calculated using the Expasy
ProtParam tool). LePLD
α
1
and
CmPLD
α
2
), and cucumber (
CsPLD
α
α
1, CmPLD
α
1, and CsPLD
α
1 are the most acidic and have the
α
α
lowest, nearly identical pIs. FaPLD
1 and LePLD
2 are intermediate in acidity and pI,
whereas LePLD
2 are only weakly acidic and have the highest, closely
similar pIs. In addition, although all seven PLD alphas were shown to be soluble pro-
teins, hydropathy calculations indicated some variation in grand average hydropathicity,
CmPLD
α
3 and CmPLD
α
α
1 > CsPLD
α
1 > LePLD
α
1 having the most negative values, and LePLD
α
2
>LePLD
2 having the least negative values.
A PESTfind search (http://bioweb.pasteur.fr/seqanal/interfaces/Pestfind-simple.html)
was conducted to determine potential proteolytic targeting sequences that are hydrophilic
spans of 12 or more amino acids (AAs) including at least one proline, one glutamic or
aspartic acid, and one serine or threonine residue, flanked by lysine, arginine, or histidine
residues (Rogers et al., 1986). The search revealed one such sequence common to all seven
PLDs in the range of AAs 628-649 (Fig. 9.12). The PEST score, considered as an inverse
measure of the protein's half-life, was highest for LePLD
α
3 > FaPLD
α
1
=
CmPLD
α
3
(6.54), and intermediate for the remaining five PLD alphas (ranging from 10.27 to 13.59).
A second PEST sequence with a much lower score was detected for both FaPLD
α
2 (17.35), lowest for LePLD
α
α
1 (2.08)
and CsPLD
1 (0.21), whereas a second sequence (AAs 605-614) with a significant score
of 14.30 was identified for CmPLD
α
α
1.
9.6.2 Secondary structure, interactions, and modulation of fruit PLD alphas
The predicted nature of PLD alphas as soluble proteins with no membrane spanning domains
suggests interesting secondary and tertiary structural and conformational characteristics that
account for membrane association as well as binding of lipid substrates and modulators.
Reverse position-specific (RPS) BLAST searches for conserved domains on the NCBI
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