Journal of Bacteriology, June 1999, p. 3705-3709, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Modes of Action of Five Different Endopectate
Lyases from Erwinia chrysanthemi 3937
Caroline
Roy,1,2
Harry
Kester,1
Jaap
Visser,1
Vladimir
Shevchik,2
Nicole
Hugouvieux-Cotte-Pattat,2
Jeanine
Robert-Baudouy,2 and
Jacques
Benen1,*
Molecular Genetics of Industrial
Micro-organisms, Wageningen Agricultural University, 6703 HA
Wageningen, The Netherlands,1 and
Laboratoire de Génétique Moléculaire des
Micro-organismes et des Interactions Cellulaires, UMR-CNRS 5577, INSA, 69 621 Villeurbanne Cedex, France2
Received 23 November 1998/Accepted 17 April 1999
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ABSTRACT |
Five endopectate lyases from the phytopathogenic bacterium
Erwinia chrysanthemi, PelA, PelB, PelD, PelI, and PelL,
were analyzed with respect to their modes of action on polymeric and
oligomeric substrates (degree of polymerization, 2 to 8). On
polygalacturonate, PelB showed higher reaction rates than PelD, PelI,
and PelA, whereas the reaction rates for PelL were extremely low. The
product progression during polygalacturonate cleavage showed a typical
depolymerization profile for each enzyme and demonstrated their
endolytic character. PelA, PelI, and PelL released oligogalacturonates
of different sizes, whereas PelD and PelB released mostly unsaturated
dimer and unsaturated trimer, respectively. Upon prolonged incubation, all enzymes degraded the primary products further, to unsaturated dimer
and trimer, except for PelL, which degraded the primary products to
unsaturated tetramer and pentamer in addition to unsaturated dimer and
trimer. The bond cleavage frequencies on oligogalacturonates revealed
differences in the modes of action of these enzymes that were
commensurate with the product progression profiles. The preferential products formed from the oligogalacturonates were unsaturated dimer for
PelD, unsaturated trimer for PelB, and unsaturated tetramer for PelI
and PelL. For PelA, preferential products were dependent on the sizes
of the oligogalacturonates. Whereas PelB and PelD displayed their
highest activities on hexagalacturonate and tetragalacturonate, respectively, PelA, PelI, and PelL were most active on the octamer, the
largest substrate used. The bond cleavage frequencies and reaction
rates were used to estimate the number of subsites of each enzyme.
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INTRODUCTION |
Erwinia chrysanthemi is a
plant pathogenic enterobacterium which causes soft-rot disease. This
bacterium secretes various enzymes that permit the degradation of
pectin, which is present in the middle lamella and primary plant cell
walls. Pectin is a complex carbohydrate consisting of smooth
homogalacturonan regions, where the backbone is partially
methylesterified and/or acetylated (4), and of the so-called
hairy regions, consisting of alternating galacturonate-rhamnose
stretches. To the rhamnose residues, side chains like arabinans or
galactans can be attached (17). To date, pectin
methylesterase, pectin acetylesterase, pectate lyase, pectin lyase, and
polygalacturonase activities have been detected in E. chrysanthemi (1, 6, 19). Among those activities, the
pectate lyases appear to be the most abundant, and they have been shown
to play an important role in soft-rot disease (2). The
pectate lyases cleave internal glycosidic bonds in polygalacturonate by
-elimination, releasing both saturated and unsaturated
oligogalacturonates. The pectate lyase activity of E. chrysanthemi results from the combined action of numerous pectate
lyases, including five major enzymes, PelA, PelB, PelC, PelD, and PelE
(23), and five minor enzymes, PelX (3, 21), PelL
(10, 18), PelZ (14), PelI (20), and
PelW (22). All pectate lyases are extracellular and have an
endo mode of action, except for PelW and PelX, which are localized in
the cytoplasm and the periplasm, respectively, and have an exolytic
activity (21, 22).
With three-dimensional (3D) structures now available for three pectate
lyases, namely, Plc and Ple from E. chrysanthemi EC16 (9, 24, 25) and Pel from Bacillus subtilis
(13), we have addressed the mode of action of five pectate
lyases to understand their specificity as determined by the nature and
number of subsites. PelA, PelB, and PelD represent the acidic, neutral,
and basic pectate lyases from family 1, respectively (5),
and PelI and PelL are representatives of families 3 and 4, respectively
(20). The enzymes were studied with respect to bond cleavage
frequencies and reaction rates on oligogalacturonates with degrees of
polymerization (n) of 2 to 8 and with respect to the
reaction rates and the product progression on polygalacturonate. This
provides a quantitative basis for their different modes of action and
allows an estimation of the number of subsites.
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MATERIALS AND METHODS |
Abbreviations.
The abbreviations used are as follows: Pel,
pectate lyase; (GalpA)n,
oligogalacturonate with indicated degree of polymerization
(n); u(GalpA)n,
4,5-unsaturated oligogalacturonate with indicated degree of
polymerization (n); HPAEC, high-performance anion-exchange
chromatography; PAD, pulsed amperometric detection.
Overproduction of the pectate lyases.
The bacterial strains,
plasmids, and techniques used to overproduce and purify the five
pectate lyases examined in the present study were described previously
(18, 20, 23). Protein concentrations were determined by the
Bio-Rad protein assay with bovine serum albumin as a standard.
Substrates.
The oligogalacturonates of defined length
(n = 2 to 8) were prepared as described previously
(7). Polygalacturonate (degree of polymerization, 150 [8]) was obtained from U.S. Biochemical Corp.
(Cleveland, Ohio).
Enzymatic assays.
Activities on polygalacturonate and
oligogalacturonates were determined spectrophotometrically by measuring
the change in absorbance at 235 nm due to the formation of
4,5-unsaturated products (
235 = 4,600 M
1 · cm1 [11]). All
assays were conducted at 37°C in 0.5 ml with 2.5 g of
polygalacturonate liter1 or 0.5 mM oligogalacturonate
(n = 2 to 8) in 20 mM buffer-1 mM CaCl2.
The buffers used were Tris-HCl (pH 8.0) for PelA, PelD, and PelL and
2-amino-methyl-propanol-HCl (pH 9.0) for PelB and PelI. These buffers
were chosen after determination of the pH optima. One unit of pectate
lyase activity is the amount of enzyme which liberates 1 µmol of
product per min.
Mode of action.
For the determination of the bond cleavage
frequencies on oligogalacturonates (n = 2 to 8) and for
the analysis of the products released from polygalacturonate, enzyme
reactions were performed under conditions identical to those described
for the enzymatic assays: 2.5 g of polygalacturonate
liter1 or 0.5 mM oligogalacturonate (n = 2 to 8), 1 mM CaCl2, and 20 mM Tris-HCl (pH 8.0) (for
PelA, PelD, and PelL) or 2-amino-methyl-propanol-HCl (pH 9.0) (for
PelB and PelI).
To determine the bond cleavage frequencies, enzyme amounts were chosen
to minimize the risk of secondary cleavage product formation. Reactions
were stopped by lowering the pH to 3.5 to 4 by the addition of 0.2 volume of 1.2% (vol/vol) acetic acid. Reaction products were analyzed
by HPAEC with PAD (Dionex Inc., Sunnyville, Calif.) and UV detection at
235 nm (Pye-Unicam LC-UV detector). The elution conditions used were
the same as those outlined previously (12). The saturated
products were quantitated from the PAD signal and the PAD response from
a calibration mixture of oligogalacturonates (n = 1 to
8) at a concentration of 0.1 mM each. The unsaturated products formed
were estimated from the peak areas of the UV profile. From the combined
data, the bond cleavage frequencies were calculated.
The product formation during polygalacturonate cleavage was studied for
up to 24 h. Normalized amounts of enzyme were added (1.80 U/ml for
PelA, 2.13 U/ml for PelB, 1.87 U/ml for PelD, 2.08 U/ml for PelI, and
1.90 U/ml for PelL). At timed intervals, 50-µl samples were withdrawn
and reactions were stopped by lowering the pH to 3.5 to 4 by the
addition of 0.2 volume of 1.2% (vol/vol) acetic acid. Reaction
products were analyzed by HPAEC as described above. The unsaturated
products formed were quantitated from the UV profiles and a standard
containing unsaturated dimer at 0.2 mM. For each enzyme, the percentage
of substrate conversion after 24 h was estimated from the combined
mass of the unsaturated products formed as quantified by HPAEC.
3D structure modelling.
In order to model the 3D structures
of PelB (accession no. X67475) and PelD (accession no. AJ132101) based
on the E. chrysanthemi EC16 Plc (PDB accession no. 2PEC) and
Ple (PDB accession no. 1PCL) structures, respectively, the Modeler
routine of the Homology module as provided by the program Insight II
(version 97.0; Molecular Simulations Inc., San Diego, Calif.) was used. The quality of the models was estimated from Ramachandran plots.
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RESULTS AND DISCUSSION |
Reaction rates and product progression on polygalacturonate.
The five enzymes studied showed very different specific activities on
polygalacturonate (Table 1). PelB and
PelD were the most active, whereas PelI and PelA showed less activity.
The activity of PelL was very low.
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TABLE 1.
Specific activities of E. chrysanthemi 3937 endopectate lyases at their pH optima with polygalacturonate as
the substratea
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The results of the product progression data for each enzyme are
presented in Fig. 1. For clarity, only
the data for the period up to 4 h of incubation are shown. Only
minor further changes were observed after 4 h. These involved
mainly a small increase in u(GalpA)2, except in
the case of PelL, which revealed a further increase in
u(GalpA)2 and u(GalpA)3
at the expense of u(GalpA)5 and
u(GalpA)6. After 24 h, 70 to 78% of the
polygalacturonate initially present, depending on the enzyme, was
converted (data not shown). For all five enzymes, the data showed the
initial formation of oligogalacturonates larger than
u(GalpA)5, which were gradually converted into
smaller products, compatible with an endolytic mode of attack of the
polymer substrate. The depolymerization profiles, which are a result of
the number of subsites, the affinity of the subsite(s) for the
substrate, and the intrinsic reaction rate, are different for all
enzymes. For PelB and PelD, one major product accumulated quickly,
namely, u(GalpA)3 and
u(GalpA)2, respectively, indicating a strong
preference for the substrate to bind in the particular mode resulting
in those products. Furthermore, for PelB, a strong accumulation of
u(GalpA)2 was also observed, although with a
lag. This suggests that smaller products bind in a different preferred
productive mode as compared to the larger products. For PelA, PelI, and
PelL, less pronounced favored products were formed. PelA converted the
initial larger products formed into u(GalpA)2
and u(GalpA)3. For PelI, a prominent transient increase in u(GalpA)4, which was degraded into
u(GalpA)2 upon further incubation was recorded.
PelL also showed accumulation of u(GalpA)4 and
u(GalpA)5, but u(GalpA)4
was not further cleaved.
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FIG. 1.
Unsaturated reaction products formed upon incubation of
polygalacturonate with the E. chrysanthemi 3937 pectate
lyases PelA (A), PelB (B), PelD (C), PelI (D), and PelL (E). Conditions
are described in Materials and Methods. The following concentrations of
enzymes were added: 1.80 U · ml1 for PelA, 2.13 U · ml1 for PelB, 1.87 U · ml1 for PelD, 2.08 U · ml1 for PelI,
and 1.90 U · ml1 for PelL. uGn,
u(GalpA)n. Symbols:  ,
u(GalpA)2;  ,
u(GalpA)3;  ,
u(GalpA)4;  ,
u(GalpA)5;  ,
u(GalpA)6;  ,
u(GalpA)7-10.
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Previously, product progression profiles were reported for Pla, Plb,
Plc, and Ple from E. chrysanthemi EC16 (15). Pla,
Plc, and Ple are most closely related to PelA, PelB, and PelD of
E. chrysanthemi 3937, respectively, with 29 amino acid
substitutions for Pla, 27 amino acid substitutions for Plc, and 22 amino acid substitutions and 7 amino acid insertions for Ple.
Surprisingly, despite these mutations, the product progression profiles
for PelA and PelD closely resemble those for Pla and Ple
(15). However, for PelB, a strong accumulation of
u(GalpA)2, which was in contrast to the profile
for Plc, was observed. The origin of these similarities and differences
will be discussed below.
Reaction rates and bond cleavage frequencies on oligogalacturonates
(n = 2 to 8).
In order to understand the origin
of the differences between the individual enzymes in product
progression on polygalacturonate and their mode of action, the turnover
rates of oligogalacturonates (n = 2 to 8) and
corresponding bond cleavage frequencies for each enzyme were studied.
None of these endo-acting enzymes showed degradation of
(GalpA)2, which is also reflected in the product
progression profiles where u(GalpA)2 accumulated
as the smallest product. Another major product observed in the product
progression profile was u(GalpA)3. The fact that
this product accumulated must be attributed to the very low conversion
rates of this substrate by all five enzymes, as revealed in studies
using unsaturated oligomers as substrates (data not shown). This is
also corroborated by the observed low reaction rates on
(GalpA)3 for all five enzymes (Table
2). Thus, the enzymes require substrates
with an n of >3 for effective catalysis.
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TABLE 2.
Bond cleavage frequencies and specific activities of the
E. chrysanthemi 3937 endopectate lyases PelA, PelI, PelL,
PelD, and PelB acting on oligogalacturonates of
defined lengthsa
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For PelA, two preferred cleavage positions were observed depending on
the size of the oligogalacturonate. Exclusive cleavage at the second
bond from the reducing end for (GalpA)3 and
(GalpA)4 and preferential cleavage at this bond
for (GalpA)5 and (GalpA)6 were found, whereas (GalpA)7 and
(GalpA)8 were preferentially cleaved at the
second bond from the nonreducing end. This shift of the preferred bond
cleaved, together with the observation that the penultimate bond from
both ends was preferred over more central bonds for oligogalacturonates
with an n of >5, strongly indicates that the number of
subsites with the highest affinity for homogalacturonan is four,
stretching from positions 
2 to +2. This is further substantiated by
the lower cleavage rates for (GalpA)5 and
(GalpA)6 relative to that for
(GalpA)4. The increased reaction rate for
(GalpA)7 and (GalpA)8
indicates that the actual number of subsites exceeds four.
Despite a 100-fold difference in reaction rates between PelI and PelL,
both enzymes showed their highest activities on the longer
oligogalacturonates (n 
6) and displayed comparable
bond cleavage frequencies, with a preference for the fourth glycosidic linkage from the reducing end. Subsequent conversion of the smaller oligomers that were formed proceeded for both enzymes at a much lower
rate, particularly for PelL, and with different end products. For PelI,
u(GalpA)4 was still a good substrate, explaining
the decrease of this product in the progression profiles. Due to the relatively much lower activity of PelL on
(GalpA)5 and (GalpA)4, these substrates are not further converted as long as
oligogalacturonates with a degree of polymerization of >5 are present.
For the smaller oligogalacturonates, the preferred cleavage was
observed at the nonreducing end for PelI whereas PelL preferred the
second glycosidic bond from the reducing end. Since the reaction rates
for PelI and PelL increased with oligogalacturonates with an
n up to 7 with increasing preference for the fourth
glycosidic linkage from the reducing end, it can be concluded that for
both enzymes the minimum number of subsites is seven, stretching from
positions 3 to +4. It should be pointed out that in view of the very
low activity of PelL, but its pronounced macerating activity
(20), homogalacturonan may not be the natural substrate.
PelD was the most prominent enzyme with respect to the preferred bond
cleaved. For oligogalacturonates with an n of 6, hardly any
products other than u(GalpA)2 were detected, and
for those with an n of 7 or 8, the second bond from the
reducing end was still strongly preferred. Thus, the bond cleavage
frequencies explain the product progression well. The strong preference
of PelD for the second glycosidic linkage from the reducing end and the
highest activity on (GalpA)4 strongly suggest
that the array of four subsites stretches from positions 2 to +2.
For PelB, cleavage of oligogalacturonates with an n of >4
occurred predominantly at the third glycosidic bond from the reducing end whereas u(GalpA)2 was formed mainly from
(GalpA)4 and (GalpA)3. The highest reaction rate was observed on
(GalpA)6. In combination with the predominant
cleavage of oligogalacturonates with an n of >4 at the
third linkage from the reducing end, this indicates the presence of six
subsites extending from positions 3 to +3.
As mentioned above, the product progression for PelB differed from that
of Plc, the E. chrysanthemi EC16 counterpart, mainly with
respect to u(GalpA)2 formation. A comparison of
the data for PelB with those obtained for Plc under slightly different conditions (16) reveals only small changes with respect to
bond cleavage frequencies and even a stronger preference for Plc to form u(GalpA)2 from
(GalpA)4 (75%). However, the rate at which (GalpA)4 is cleaved by Plc relative to that of
the larger oligogalacturonates is lower than the same cleavage ratio
for PelB
[(GalpA)4/(GalpA)6 cleavage ratios for Plc and PelB are 1 to 4.6 and 1 to 1.7, respectively]. This indeed can account for the observed difference in
product progression.
Since PelB and Plc differ with respect to only 27 of 353 amino acids,
the PelB 3D structure was modeled by using the Plc 3D structure
(16). Most amino acid substitutions took place at a site
that was remote from the substrate binding cleft (16) in the
N-terminal part of the enzyme that forms an 
-helix capping the
-helix and at the surface of the enzyme. Those mutations are,
therefore, probably not involved in enzyme specificity. However, three
amino acid substitutions (G161N, A167S, and S239G; the last residues
refer to those of Plc) were found in the substrate binding cleft. At
least one of the substitutions, S239G, may involve (in)direct contact
with the substrate due to the change of the functionality of the side
chain. In the modeled PelB structure, the G161N mutation results in a
changed conformation of D160. In the Plc-substrate structure, D160 and
D162 coordinate a Ca2+ ion that is in contact with the
substrate (16). The A167S mutation most probably does not
affect the enzymatic properties since its position below the
Ca2+ ion present in both the substrate-free enzyme and the
enzyme-substrate complex is not likely to influence the
Ca2+ ion that is coordinated by the four conserved acidic
residues D129, D131, E166, and D170. Thus, the observed changes in the modeled 3D structure of PelB, notably G161N and S239G, may well account
for the different product progression profiles of PelB and Plc.
The limited number of amino acid differences observed for PelD and Ple
also allowed the modeling of the PelD 3D structure based on the Ple 3D
structure (9). None of the amino acid substitutions was
found to occur in the substrate binding cleft as inferred from the
Plc-substrate complex (16). The similar product progression profiles for PelD and Ple and those differing for PelB and Plc indicate
that the naturally occurring mutations do not change the catalytic
properties unless they occur in the substrate binding cleft. From this,
it logically follows that the amino acid differences between PelA and
Pla are most likely in positions remote from the substrate binding cleft.
For all of the enzymes studied here, the bond cleavage frequencies and
substrate reaction rates on oligogalacturonates (n = 2
to 8) easily explain the product progression on the model substrate
polygalacturonate. Still puzzling are the much higher activities of
PelB and PelI on oligogalacturonates relative to those on
polygalacturonate. A plausible explanation would be that the polymer
substrate obviously extends beyond the array of subsites and that this
hampers its effective binding.
In physiological terms, PelD, PelB, and PelI, being the most active
enzymes and generating u(GalpA)2,
u(GalpA)3, and u(GalpA)4, respectively, might serve as the major substrate supply for the bacterium. Both u(GalpA)2 and
u(GalpA)3 may be internalized directly (6). PelX might cleave u(GalpA)4 into
u(GalpA)2 (22).
It has been shown that PelD and PelI are very active in tissue
maceration, that PelB and PelL are moderately active in this respect,
and that PelA is virtually devoid of this activity (1, 10,
20). So far, we have not been able to establish a relationship between specific activity, mode of action, and macerating properties of
the enzymes. Further elucidation of the substrate specificity of the
pectate lyases must await the isolation of well-defined complex
substrates and the elucidation of 3D structures of enzyme-substrate complexes.
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ACKNOWLEDGMENT |
This work was supported by E.C. grant AIR2-CT941345 to J.V. and
J.R.-B.
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FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Genetics of Industrial Micro-organisms, Wageningen Agricultural
University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands. Phone:
31 317484439. Fax: 31 317484011. E-mail:
jac.benen{at}algemeen.mgim.wau.nl.
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Journal of Bacteriology, June 1999, p. 3705-3709, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
