From the Unité Mixte de Recherche Centre
National de la Recherche Scientifique 7632, Université P. et M. Curie, case 154, 4 Place Jussieu, 75252 Paris Cedex 05, ¶ Centre
de Recherches sur les Macromolécules Végétales,
Centre National de la Recherche Scientifique (associated with
University Joseph Fourier), BP 53, 38041 Grenoble Cedex 9, and
** Service de Coopération Université/Entreprises/Organismes
Régioneaux Centre National de la Recherche Scientifique
Unité Propre de Recherche de l'Enseignement Supérieur
Associée 203, Faculté des Sciences, Université de
Rouen, 76821 Mont-Saint-Aignan Cedex, France
Received for publication, September 29, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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Well-characterized pectin samples with a wide
range of degrees of esterification (39-74%) were incubated with the
solubilized pure Pectinmethylesterases
(PMEs)1 are cell wall-bound
proteins present in almost all plants and phytopathogenic
microorganisms. They modify pectic homogalacturonan chains, generating
free carboxyl groups along the polygalacturonan backbone and releasing
protons into the apoplasm. Their action can therefore lead to
antinomical effects, especially in primary cell walls. On one hand, the
pH decrease should enhance expansin activity and in turn increase the
cell wall extensibility (1), but, on the other hand, the generation of
carboxyl blocks would be expected to allow the formation of multichain
structures via calcium bridges (2). Such structures would greatly
affect the physical properties of pectin, due to the assembly of pectic
chains into expanded, highly hydrated gel networks decreasing cell wall
porosity and also cell wall extensibility (3). Such Ca2+
bridging requires the occurrence of nearly 10 successive carboxyl groups (4) and implies that the PMEs work processively along the
galacturonan chain. Unfortunately, although PME biochemical and
molecular characteristics have been widely investigated (5-8), a
limited number of studies of their action pattern have been reported
(9-13). Recently, using partially depolymerized pectin samples and
different PME isoforms extracted from mung bean hypocotyl cell walls,
we (14) determined both enzyme activity and average product structure
at regular intervals along the deesterification pathway. Simulations of
different mechanisms by fitting to the experimental data provided
information on the possible action patterns of three isoforms. In the
case of two of them, the so-called PE In the present study, we set out to further investigate the effects of
neutral and acidic pHs on the development of the deesterification process catalyzed by the neutral and alkaline PME isoforms (PE Pectin Samples--
The three pectin samples PS1, PS2, and PS3,
which differed in their DE, were graciously supplied by Herbstreith & Fox KG (Neuenbürg, Germany).
Enzyme Isolation and Assay--
Cell walls were isolated from
the upper 2.5 cm of hypocotyl tissues of 3-day-old seedlings of mung
bean (Vigna radiata L. Wilzeck) according to a procedure
described previously (15). PMEs bound to the cell wall fragments were
solubilized with 1 M NaCl, and the different isoforms were
recovered as described by Bordenave and Goldberg (16, 17). PME activity
was measured titrimetically by following the release of protons from
the pectin sample in the presence of 150 mM NaCl (total
volume of the assay, 6 ml). The protons were titrated with 10 mM NaOH under nitrogen, the pH was maintained at pH 5.6 or
pH 7.6 with an automatic titrator (TTT 80; Radiometer), and the
reaction rate was expressed as µeq H+ released/min. The
reaction was stopped by lowering the pH to pH 3 with 0.05 N HCl.
Interaction Experiments--
To check whether some differences
could be detected in pectin behavior after action of PE Simulations--
Deesterification simulations were conducted
with in-house software as described previously (14). Starting samples
consisting of 1,000 chains of 100 residues with a Bernoullian (random)
distribution of methylester groups and DE values of 73% (PS1), 53%
(PS2), and 38% (PS3) were constructed with a random number generator
(E, methylesterified galacturonic acid residue; U, galacturonic acid residue with a free carboxyl group; X, unspecified residue
that may be E or U; E, residue in the process of
deesterification). PME deesterification was simulated as random attack
followed by blockwise deesterification according to the SCM or MAM that
included constraint criteria. The latter feature took into account the requirement that a free carboxyl group (U) be located 2 residues away
from the point of attack (UXE constraint). At regular
intervals along the deesterification pathway (DE steps of 5%), average
populations such as the number of blocks of various lengths were
determined. To assess the influence of small variations in DE
(~1-2%) on the block size of the largest U block, data were also
simulated five times with different initial seeds at DE
intervals of 1% for the SCM with the UXE constraint.
Simulations were repeated at least five times with different initial
seeds for the random number generator, and the average values
for the U block populations for the single-chain and multiple attack
mechanisms were determined.
Reverse Transcription-PCR and cDNA Cloning of the Isoform
PME Characteristics of the Pectin Samples Used in the Assays--
The
pectin samples used in the assays have been described previously (22).
The three samples (PS1, PS2, and PS3) had average molecular
masses of 188, 148, and 154 kDa and DEs of 74%, 54%, and 39%,
respectively. These pectins presented some side chains (9%, 5%, and
5%, respectively), and the methoxyl distribution of PS1 and PS2 was
random, whereas that of PS3 was slightly blockwise. This latter
information could be reliably obtained from 13C NMR spectra
because it was possible to establish triad populations (i.e.
the relative fractions of EEE,
EEU+UEE, and UEU trimers within the
polymer could be evaluated by integration of the C6 signals). The
percentages of methylester groups suitable for PME binding in PS1, PS2,
and PS3 (i.e. satisfying the UXE constraint) were
estimated to be 27%, 46%, and 60%, respectively, from the UXE populations of the theoretical pectins.
Activity of PME Isoforms toward Pectins with Different
DEs--
The kinetic parameters of the two PME isoforms, PE Interaction Experiments--
Preliminary data indicate that upon
incubation with PE Time Course of Enzymatic Deesterification of Pectins with Different
DEs--
To visualize the effects of DE on the time course of the
reaction rate of the deesterification process, plots of activity versus DE have been traced for pectin samples differing in
their DE (Fig. 1). Incubations were
performed either at pH 5.6 or pH 7.6. The plots obtained with the two
isoforms differed, but whatever the incubation conditions, none of the
enzymatic fractions were able to generate totally deesterified pectins,
and the final DE depended on the nature of the substrate. At pH 7.6, with the neutral isoform PE Deesterification Limits--
Assays were performed to identify the
factors responsible for the drop in activity at both pHs that were
investigated. They consisted of attempts to restore the activity at the
end of the deesterification process either by adding new substrate or
enzyme molecules in the incubation medium or by changing the pH.
Addition of Enzymes--
Whatever the pH, the addition of new
enzyme molecules at the end of the PE Addition of Substrate--
PE Modification of the pH--
PE Effect of Galacturonic Units--
At pH 5.6, with both isoforms,
addition of PGA in the assay induced a decrease in the velocity.
Preliminary experiments carried out with PE Simulations--
The numerical simulations of the U block
populations during deesterification by either SCM or MAM requiring a
free carboxyl group at the second nearest neighbor position
(UXE constraint) were based on five runs with 1,000 chains
of polymer (PS1, PS2, or PS3 with chain lengths of 100 residues).
Turnover of pectin during the deesterification process has been
evaluated to be 300-400 molecules (or successful pectin/PME
encounters)/enzyme molecule/s from the specific activity (20) and the
molar mass and this turnover was in agreement with turnovers
reported for orange PMEs (10). Considering that the substrate:enzyme
ratio in the activity assays was roughly the same as that in the
simulations (1,000:1), such a turnover implies that as soon as a single
pectin with a U block sufficiently long to cause inhibition of the
enzyme is formed, a change of slope should be observed in the activity
profiles in Fig. 1.
The sampling in the numerical simulations is too small to give
statistical populations and the standard deviations for significant populations are in the 5-10% range (i.e. 12-residue U
blocks of PS2 at DE 28% in the MAM simulations; Table III, 221 ± 9 chains). In the case of the smallest populations, these standard
deviations are close to 100% (i.e. the 19-residue U blocks
in the SCM simulation; Table II, at the slope change points of PME
activity at pH 5.6 in Fig. 1 (PS1, 4 ± 1.5; PS2, 1.5 ± 0.5;
PS3, 1 ± 1.3)). Thus, the block lengths with the smallest
populations at the change of slope points for the incubations with
PE
It has been shown that the SCM requiring a free carboxyl group at the
next nearest neighbor position (UXE constraint) reproduces the residue distributions (primary sequence of U and E residues) all
along the deesterification pathway for both PE
At pH 7.6, a MAM with the UXE constraint has been
demonstrated for PE Comparison of PE
Comparison of the partial PE It was demonstrated previously (14) that PE However, a comparison of the curves in Fig. 1 for PS1, PS2, and PS3
reveals new paradoxes. Indeed, both enzymes show enhanced activity for
a native substrate with a random distribution of carboxyl groups as
opposed to a pectin of identical DE that has undergone enzymatic
deesterification, leading to a blockwise distribution of pendant
groups. The greater activity with the former substrates undoubtedly
reflects the higher probability of satisfying the required
UXE condition for pectin binding when these polar groups are
randomly distributed along the pectic chain rather than localized in
acidic blocks.
The present study has also shown through the inhibition assays that
chains with long U blocks such as polygalacturonic acid are competitive
inhibitors of PE At pH 7.6, inhibition of PE At the molecular level, the explanation for the pH effect is still
difficult to assess. Differences in the kinetic parameters for PE The results described in the present study underline the importance of
the distribution of the carboxyl units along the pectin backbone in the
enzymatic deesterification of pectins. This parameter controls the
activity of the cell wall PMEs to a much greater extent than the
methylesterification degree, with a random distribution being the most
favorable situation. Moreover, it has been clearly demonstrated that
whatever the pH or the enzyme used in the assay, the deesterification
process progressively drops to a negligible level, even though the
pectins are far from being totally deesterified. This observation can
explain the coexistence in most cell walls of methylesterified pectins
and active PMEs. Considering that the cell wall pH is around
5.6, irregardless of the PME isoforms present in planta, it
can be predicted that the enzymatic deesterification will stop abruptly
after giving rise to U blocks of roughly 20 residues. The occurrence of
relatively short acidic blocks interspacing the pectic backbone has
already been suggested (35). Calcium ions, which are abundant in the
cell walls, will then induce the formation of junction zones, giving
rise to a gel network. Recent data (36) showed that in mung bean
hypocotyl, calcium ions are present mostly in elongated cell walls and
colocalized with acidic polygalacturonan blocks in tricellular
junctions. The formation in precise cell wall domains of multichain
structures will modify the cell wall cohesion and restrict
extensibility. The feedback control of the demethylesterification might
represent a kind of protection because totally deesterified
homogalacturonan could disturb the apoplasmic traffic. Occurrence
inside the cell walls of microdomains is now documented (37), and the
localization in precise areas of acidic pectin blocks results in part
from the specific mechanisms of the enzymatic demethylesterification process. Further investigation of cell wall rheological properties of
transgenic plants with reduced PME activity might provide useful information for a better and more precise understanding of PME functions in planta.
and
isoforms of pectinmethylesterase, from
mung bean hypocotyl (Vigna radiata). Enzyme activity was
determined at regular intervals along the deesterification pathway at
pH 5.6 and pH 7.6. It has been demonstrated that the distribution of
the carboxyl units along the pectin backbone controls the activity of
the cell wall pectinmethylesterases to a much greater extent than the
methylation degree, with a random distribution leading to the strongest
activity. Polygalacturonic acid was shown to be a competitive inhibitor of the
isoform activity at pH 5.6 and to inhibit the
isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in
enzyme activity was shown to be correlated to the formation of
deesterified blocks of 19 ± 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du
Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem.
273, 33150-33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the
substrate had been totally deesterified. Comparison of
and
isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in
this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pI around 7.5) and
PE
(pI above 9) isoforms, a single-chain mechanism (SCM) associated
with a free carboxyl group at the second nearest neighbor position was
postulated at pH 5.6, whereas some multiple attack mechanism (MAM) was
required to reproduce the experimental data at pH 7.6. Although these
action patterns reproduced the free carboxyl and methylester
distribution in pectin incubated with PE
and PE
at both pHs for
the entire degree of esterification (DE) range observed experimentally,
they did not explain the marked decrease in activity for DE values
below 50-60%. Indeed, in the simulated digestions, deesterification
occurred smoothly down to DE values between 2-4%.
and
PE
, respectively). We wanted to determine what limits the deesterification process in situ because, in young cell
walls, active PMEs coexist with highly methylated pectins. Three
well-characterized commercial pectin samples differing in their DE were
chosen for these investigations. We endeavored (a) to
determine the kinetic constants of deesterification, (b) to
establish the activity profiles as a function of the deesterification
pathway for all three samples, (c) to check both
experimentally and with simulations possible limiting factors such as
inhibition by the acidic blocks produced during the reaction,
(d) to evaluate the lengths of such acidic blocks through
simulations, and (e) to compare the cDNA-deduced peptide
sequences of both isoforms to understand the possible interactions
required for the stabilization of pectin-PME binding at pH 5.6 and pH
7.6. The information obtained with this approach was expected to shed
light on the role of the different PME isoforms in the cell wall
stiffening process that occurs along the mung bean hypocotyl.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
at either pH
5.6 or pH 7.6, interaction experiments (18) were run as follows.
Pectins with a DE of 74% were treated with PE
as described above
until their DE reached 50%, and then HCl was added rapidly to shift
the pH value to pH 3, inactivate the enzyme, and facilitate the
precipitation of pectins with ethanol. After several cycles of
precipitation/solubilization in deionized water, pectins (cleansed of
salts) were finally dissolved in water at a concentration of 10 mg/ml,
corresponding to about 2.5 mM free galacturonic acid.
Increasing amounts of calcium chloride were added to the solutions, and
pectins were allowed to precipitate. To evaluate the interaction of
pectins with calcium ions, the amount of residual sugars was determined
colorimetrically in the supernatant after centrifugation at 10,000 × g for 15 min, according to the method of Dubois et
al. (19).
--
PCR was used to amplify the coding region of PE
using
degenerate primers that were designed with regard to internal peptides of the protein, as described previously (20). Total RNA was prepared
from 3-day-old mung bean seedlings according to the guanidinium thiocyanate method (21) and used to synthesize cDNA (first-strand cDNA synthesis kit; Amersham Pharmacia Biotech). Using primers prPEW (sense 5'-AGGNGCNTAYTTYGARAA) and prPEZ (antisense
5'-ANCKNCCYTGNGCNGTRAA), which code for the peptide sequences GAYFEN
and FTAQGR of two internal peptides, respectively, a 450-base
pair fragment was obtained using cDNA as template. PCR was
performed using Taq DNA polymerase (Appligene) with the
following temperature profile: 1 min at 94 °C, 1 min at 50 °C,
and 2 min at 70 °C, for 35 cycles. The purified PCR fragment was
cloned into pMosBlue vector (Amersham Pharmacia Biotech).
Upon sequencing, this fragment was shown to include the coding regions
of several internal peptides. The 3'-/5'-rapid amplification of
cDNA ends system (Life Technologies, Inc.) was then used to obtain
the full-length cDNA of PME
. For each cloned PCR product, three
independent clones were sequenced on both strands using the T7
Sequenase Quick denature plasmid sequencing kit (Amersham Pharmacia
Biotech). A cDNA library was also constructed in
ZAP II
(Stratagene) from polyadenylated RNA of mung bean hypocotyls using
XL1-Blue cells as indicated by the manufacturer. The PCR fragment was
labeled with digoxigenin (Roche Molecular Biochemicals) and used to
screen the cDNA library.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
PE
, acting on various pectin samples differing in their
methylesterification degrees have been collected in Table
I. With both enzyme fractions and at pH
5.6 as well as at pH 7.6, the lower the DE, the higher the affinity.
When the Km was calculated for the esterified residues suitable for enzyme binding, i.e. the esterified
residues satisfying the requirement that a free carboxyl group be
located at the second nearest neighbor position (UXE
constraint), the affinity also increased when the DE decreased.
However, these increases were smaller than those calculated for the
total methylester populations. With regard to the effects of pH, with
the neutral isoform, PE
, whose activity is known to be modified
relatively little by the ionic conditions (pH or saline concentration
(20)), the kinetic parameters (Vmax and
Km) were rather similar at pH 5.6 and pH 7.6. In
contrast, for PE
, both the affinity and the maximal velocity were
higher at pH 5.6, whatever the DE of the substrate.
Kinetic parameters of PE and PE
and PE
measured at pH 5.6 and pH
7.6 in 150 mM NaCl are given for various pectin samples:
PS1, DE = 74%; PS2, DE = 54%; PS3, DE = 39%.
Vmax is given as µmol of H+ min/µg, and
Km is given as mM methylester groups,
taking into account either the total concentration of methylester
groups (1) or the concentration of methylester groups suitable for PME
binding (2), estimated as described under "Material and Methods."
, pectins formed a gel that can be pelleted only
at CaCl2 concentrations higher than 0.5 mM.
Moreover, in the presence of 2.5 mM or more calcium ions,
80 ± 3% of the pectins sedimented at pH 5.6 and only 72 ± 4% of the pectins sedimented at pH 7.6. These results are in agreement
with those of Penel et al. (18), who conducted similar
experiments with polygalacturonic acid (PGA). The differences, although
small, were observed repeatedly, whatever the concentration of
CaCl2 (from 0.5 to 5 mM), indicating a
difference in the distribution of the negative charges produced by the enzyme.
, two successive phases were observed
(Fig. 1A): during the first phase, the reaction rate was
nearly constant, whereas during the second one, it decreased rapidly.
The DE value that corresponds to the greatest change in slope of
the activity profiles is referred to as the slope change point
(i.e. SCPE
or SCPE
in Tables
II and III)
and is indicated with an arrow in Fig. 1. At pH 5.6, this
second phase predominated because the activity dropped very rapidly.
The plateau observed at pH 7.6 was much shorter with PE
and
decreased with DE (Fig. 1B). With both isoforms, for a given
DE, the reaction rate depended strictly on the nature of the pectin
substrate. The reaction rate was always much lower when the DE resulted
from enzymatic deesterification (e.g. the activity of PE
with PS1 at a DE of 53% as compared with that with PS2 at the
beginning of the reaction). According to previous observations (14),
both isoforms produce acidic blocks along the pectin chains at a rate
that depends on the pH. In contrast, the native pectin samples
are characterized by a random distribution of the acidic units (22).
These data indicate that the distribution of the carboxyl and methoxyl
groups along the polymer backbone is an important factor for the
activity, with a random distribution of the carboxyl units inducing in
all cases a higher activity than a blockwise one.
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Fig. 1.
Development of PME activity during the
deesterification process of various pectin samples by
PME (A) and
PME
(B). The pH of the
incubation medium was maintained at either pH 5.6 (filled
symbols) or pH 7.6 (open symbols). The activity (in
µeq of H+ released/min/µg protein) was estimated from
the time necessary to release 2.5 µeq of H+ from the
pectin sample. For each enzyme, the initial reaction rate for DE
70-75% at pH 5.6 has been normalized to 100. Circles
represent the deesterification of PS1, triangles represent
the deesterification of PS2, and squares represent the
deesterification of PS3. The DE values that correspond to the greatest
change in slope of the activity profiles are referred to as the slope
change points (i.e. SCPE
or SCPE
in Tables II and III)
and are indicated by filled (pH 5.6) and open (pH
7.6) arrows.
Distribution of U blocks during digestion by SCM
(SCPE
)a and PE
(SCPE
).a
Distribution of U blocks during digestion by MAM
(SCPE
)a and PE
(SCPE
).a
-catalyzed deesterification did
not allow resumption of the enzymatic reaction (Fig.
2), indicating that the modified substrate generated upon PE
action cannot be further processed.
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Fig. 2.
Addition of new enzyme molecules at the end
of the deesterification process represented as a plot of activity
(µeq of H+ released)
versus incubation time. PME was incubated with
the pectin sample PS1 (DE 74%) at pH 5.6 (
) or pH 7.6 (
). At the
arrows, new enzyme molecules were added in the
assay.
was incubated either at pH 5.6 or
at pH 7.6 with PS1 (DE, 74%), and the reaction was allowed to progress
until the rate became negligible (Fig.
3). PS1 in NaCl was then added to the assay so that the initial conditions were restored. At pH 7.6, the
deesterification started again and developed as seen initially. In
contrast, at pH 5.6, the reaction started again but progressed much
more slowly than it did initially (initial velocity was only a third of
that developing at the beginning of the experiment). In this case, the
presence in the assay of the enzymatically deesterified substrate,
i.e. the presence of acidic blocks, inhibited the
deesterification of the native pectin sample. Similar observations were
made for PE
at acidic pH (data not shown) but at pH 7.6, contrary to
PE
, the addition of pectin induced no or only a very attenuated
resumption of the deesterification process (Fig. 3).
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Fig. 3.
Addition of new pectin molecules at the end
of the deesterification process represented as a plot of activity
(µeq of H+ released)
versus incubation time. PME
(circles) or PME
(triangles) was incubated
with PS1 (DE 74%) at pH 5.6 (filled symbols) or pH 7.6 (open symbols). At the arrows, new pectin
molecules (concentration of the methylester bonds = 18.6 mM, similar to that present at the beginning of the assay)
were added.
was incubated at pH 5.6 with PS1
until the DE was lowered to nearly 60%, and the pH was then adjusted
to pH 7.6. The deesterification started again and proceeded rather
rapidly until a DE of ~38% (Fig. 4)
was reached. In contrast, if the pH change was made when the reaction
rate had become nearly insignificant, which was achieved for a DE
around 50%, the deesterification started again but was very
short-lived (Fig. 4). However, addition at that time of new pectin
molecules induced a strong resumption of the deesterification process
(data not shown), similar to the one observed in Fig. 3.
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Fig. 4.
Modification of the pH at different steps of
the deesterification process represented as a plot of activity
(µeq of H+ released)
versus incubation time. PME was incubated with
PS1 (DE 74%) at pH 5.6. At the arrows, the pH was
adjusted to 7.6.
showed that the
inhibition was competitive with a Ki estimated to be
4.2 mM galacturonic acid residues. In contrast, at pH 7.6, the two isoforms exhibited a different sensitivity to PGA. With PE
,
even high concentrations of PGA (i.e. 7-fold the substrate
concentration) did not modify the reaction rate. At this pH, PE
activity was inhibited by PGA (i.e. 10 mM galacturonic units induced a 50% decrease of activity). With both isoforms, whatever the pH value, addition of galacturonic acid (the
monomer) was without effect. These data confirm the observations made
with respect to Fig. 3. At pH 7.6, the presence of new carboxyl groups
on the pectin substrate that resulted from enzyme action inhibits the
deesterification of new pectin molecules by PE
but not by PE
,
whereas at acidic pH, the activity of both isoforms is reduced.
(SCPE
) and PE
(SCPE
) that would be expected to cause
inhibition of enzymatic activity cannot be given with greater accuracy
than ± 1 residue. Moreover, considering the deviations of the
experimental points in Fig. 1 with respect to a smooth regular curve
with two well-defined regions (a plateau and a region with a steep
slope), these slope change points cannot be determined more precisely
than within 1% in the more favorable cases. These deviations of 1% in
DE correspond to a variation of 1 residue in the length of the largest
U block. Reasonable U block ranges that take into account these
standard deviations have been surrounded with a frame in the Tables II and III.
and PE
at pH 5.6. Indeed, common slope change points (SCPE
= SCPE
) were observed for these two enzymes with PS1 and PS2 (for DE values of 68%
and 48%, respectively). Enzymatic activity decreased regularly for
both isoforms from the beginning of the assay with PS3 (i.e. a marked plateau was not observed). For all three polysaccharides at pH
5.6, the sharp decrease in PME activity in Fig. 1 coincided with the
appearance of acidic blocks of 19 ± 1 residues in the numerical
simulations (Table II).
and PE
, but the slope change point of the
enzymatic activity occurs much further along the deesterification
pathway in the case of PE
(SCPE
corresponds to a DE of 44%,
28%, and 18%, respectively, for PS1, PS2, and PS3) when compared with
PE
(SCPE
corresponds to a DE of 66%, 48%, and 38%,
respectively, for PS1, PS2, and PS3). The longest U blocks that appear
at DE values corresponding to the SCPE
in Table III have again been framed. As for the reaction at pH 5.6, the beginning of the rapid decrease in PE
activity is concomitant with the formation of acidic
U blocks of 19 ± 1 residues in the numerical simulations. In the
case of PE
, very long U blocks are present in substantial amounts in
the theoretical deesterifications at the experimental DE values for the
slope change point at SCPE
(the longest U blocks contain between 30 and 50 residues; data not shown in Table III).
and PE
Peptide Sequences--
The
cDNA-deduced amino acid sequence of isoform PE
has been
determined previously (20). The mature PE
polypeptide is composed of
318 amino acids with a calculated molecular mass of 34.7 kDa and an
estimated pI of 9.84. These values are consistent with those observed
by SDS-polyacrylamide gel electrophoresis and isoelectric focussing (16). In the present study, we describe the
obtainment of a partial cDNA clone encoding the PE
isoform. This clone was obtained by reverse transcription-PCR using two
degenerate primers that were designated from the results of
protein sequencing (20). Attempts to get the full-length cDNA were
done using the 3'-/5'-rapid amplification of cDNA ends technique,
but only the 3'-rapid amplification of cDNA ends was successful.
Although 5'-rapid amplification of cDNA ends was performed
repeatedly on different RNA preparations and using commercial kits from
different suppliers, we were unable to get the missing 5'-end. Even the
plaque blotting method using the digoxigenin-labeled DNA fragment of
PE
to screen a cDNA library failed to give the complete
cDNA. Nevertheless, the partial amino acid sequence of PE
deduced from the cDNA clone (Fig. 5)
perfectly matches the internal peptide sequences determined previously
from the purified authentic enzyme (20). The sequence comprises 277 amino acids, with a calculated molecular mass of 30.9 kDa. Comparison of all the known PME protein sequences reveals the presence of a highly
conserved C-terminal catalytic domain with an average size of 320 amino
acids. Therefore, the partial PE
protein sequence covers a very
large part of the catalytic domain of the protein (estimated to more
than 85%). This partial PE
sequence contains four putative
N-glycosylation sites located predominantly in the second
half of the catalytic domain, whereas only one is present in the mature
PE
protein. Because the purified PE
isoform has an apparent
molecular mass of 45 kDa (16), this may signify that PE
, but not
PE
, is highly glycosylated. The partial PE
sequence has a
calculated pI of 8.34, whereas the pI of the native enzyme was
previously estimated to be 7.5 (16). Therefore, the missing N terminus
of the catalytic domain contributes greatly to the neutral pI value
observed for PE
. By comparison, the removal of the first 40 residues
of the mature PE
(Fig. 5) protein resulted in a polypeptide with a
lower calculated pI value (8.9 instead of 9.84). These observations
indicate that the N-terminal portion of the mature polypeptides confers
mainly the basic or neutral character of the two PME isoforms.
View larger version (26K):
[in a new window]
Fig. 5.
Protein sequence alignment of mung bean
PME and PME
isoforms. The regions of the protein sequence corresponding
to the internal peptides of PME
(20) are underlined.
Asterisks (*) indicate the invariant residues identified
when comparing all known PME sequences from plants, fungi, and
bacteria. Black-shaded regions correspond to the four most
conserved peptide regions found in all PMEs. The putative
N-glycosylation sites are shaded in gray.
protein sequence with that of PE
revealed 57% identity and 69% similarity. When considering all the
known PME sequences from plants, fungi, and bacteria, including PE
and PE
, 12 residues located in the four most conserved regions (Fig.
5) were found to be invariant. These residues, which include 1 arginine
residue, 2 aromatic residues, and 2 aspartate residues, are likely to
be involved in enzyme function and in maintaining a functional
conformation. In contrast, the residues that could explain the
differences in the action patterns of the PME isoforms probably reside
in more variable regions. The inhibition experiments with PGA, coupled
with the results shown in Figs. 2-4, suggest the implication of one or
more likely several basic residues in both proteins. For PE
,
residues exhibiting a pKa in the 6-7 range (only
histidine has such a pKa) are good candidates to
explain the lack of inhibition of the enzyme by PGA observed only at pH
7.6 as well as the kinetic behavior of the enzyme at this pH. At the
same position(s), arginine or lysine residues are expected to occur in
PE
. The examination of both peptide sequences did not reveal the
location of such residues, suggesting that they may reside in the
missing N terminus of PE
, the portion of the catalytic domain that
contributes greatly to the final pI of the enzyme.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PE
from
V. radiata deesterify PS1 (DE, 74%) according to the SCM
and MAM at pH 5.6 and pH 7.6, respectively, with the requirement that a
free carboxyl group be located at the second nearest neighbor position
(UXE constraint). The activity versus DE profiles
in Fig. 1 confirm this dichotomy in the action pattern with a change in
pH for substrates with a wide range of DE values (39-74%). Moreover,
results of interaction experiments suggest that the distribution of the
newly formed charges resulting from incubation at either pH 5.6 or pH
7.6 is effectively different. Higher affinity for Ca2+ in
the pectins incubated with PE
at pH 5.6 as opposed to those incubated with PE
at pH 7.6 is consistent with the formation of
longer U blocks, corroborating the more pronounced blockwise nature of
the deesterification process at lower pH.
at pH 5.6. Simulations of the deesterification
process for all three pectic samples (PS1, PS2, and PS3) with the SCM
have revealed that the slope change in both PE
and PE
activity is
concomitant with the formation of U blocks of roughly 19 ± 1 residues. It is tempting to relate this block length to either the
number of contiguous unesterified residues necessary for the formation
of aggregates or the number of residues in the binding site. The former
hypothesis is plausible because the formation of calcium linkages
between adjacent regions of 15-20 galacturonic acid residues on pectic
chains has been reported for calcium pectate gels (23). It has been
demonstrated recently by small angle x-ray scattering (24) that these
gels can correspond to bundles of 2-20 chains. It is more difficult to
examine the latter hypothesis because no crystal structures of plant
PME have yet been published. However, it should be noted that Catoire
et al. (22) have recently shown that PME can only work on
galacturonan whose degree of polymerization was around or higher
than 20, in agreement with the second hypothesis.
by PGA is observed, and this also
corresponds to the formation of U blocks of roughly 19 ± 1 residues during deesterification simulated with the MAM. In contrast, PE
is not inhibited by PGA under neutral conditions (pH 7.6), and
very long U blocks (30-50 residues) are formed before a significant loss of activity is observed. Nonetheless, the deesterification process
stops before its theoretical completion. The termination of the
reaction may be due to the lack of hydrophobic residues that might be
necessary either for binding or to diminish the substrate/solvent
interactions, thus indirectly promoting
binding.2 Indeed, in the case
of PE
, addition of substrate at pH 7.6 led to a complete resumption
of enzyme activity.
and PE
as a function of pH, as well as the observed competitive inhibition by PGA, suggest the involvement of basic residues (histidine residue(s) for PE
and arginine or lysine residue(s) for PE
) that
are presumably located in the N terminus of the catalytic domain.
Although the PME sequences show no obvious homology to other proteins,
the recent crystal structure of a PME from Erwinia chrysanthemi (25) reveals that this protein adopts the
right-handed parallel
-helix architecture that is common to other
pectinolytic enzymes such as pectin and pectate lyases (26-30),
polygalacturonases (31, 32), and rhamnogalacturonase (33). In addition,
endopolygalacturonase is also competitively inhibited by cross-linked
pectic acid (34), suggesting analogous inhibition patterns for PE
and PE
and polygalacturonase. The availability in the coming months
of the coordinates of the first crystal structure of PME (25) will be
of great value to further our understanding of the deesterification
process by PE
and PE
and will serve as a template for molecular
modeling studies.
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FOOTNOTES |
---|
* This work was supported by the Centre National de la Recherche Scientifique.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF229849 and X94443 for Vigna radiata PE and
PE
, respectively.
§ To whom correspondence should be addressed. Tel.: 33-1-44-27-59-19; Fax: 33-1-44-27-36-47; E-mail: goldberg@ccr.jussieu.fr.
Full-time investigator of Institut National de la Recherche Agronomique.
Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.M001791200
2 Peter Swarén, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PME, pectinmethylesterase; DE, degree of methylesterification; MAM, multiple attack mechanism; PGA, polygalacturonic acid; SCM, single-chain mechanism; PCR, polymerase chain reaction.
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