The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in Erwinia chrysanthemi 3937

Abstract

Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi. The E. chrysanthemi 3937 gene kdgC has been shown to exhibit a high degree of similarity to the genes pelY of Yersinia pseudotuberculosis and pelB of Erwinia carotovora, which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the kdgC gene was renamed pelW. PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca²⁺ is unable to restore the activity of PelW or Ogl, while several other cations, including Co²⁺, Mn²⁺, and Ni²⁺, can activate both cytoplasmic lyases.

The enterobacterium Erwinia chrysanthemi causes soft rot disease of various plants. Its pathogenicity is largely due to its ability to degrade pectin, a major constituent of plant cell walls and middle lamellae. The breakdown of pectin, a polysaccharide with a backbone consisting of partially esterified galacturonic acid, involves a battery of pectinases. The combination of different enzymatic activities allows E. chrysanthemi to efficiently degrade pectin and to subsequently use the liberated pectic oligomers as a carbon source for growth. The pectin esterases (pectin methylesterase and pectin acetylesterase) remove the ester groups of pectin and facilitate the action of depolymerases (pectate lyase and polygalacturonase) (12, 33). These depolymerases differ mainly in their reaction mechanisms (β-elimination or hydrolysis) and in the random or terminal mode of attack of the polymer (endo or exo).

The majority of pectinases are secreted by E. chrysanthemi into the extracellular medium through the type II secretion machinery, the Out system (35). In E. chrysanthemi 3937, eight endo-pectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ), the pectin methylesterase PemA, and the pectin acetylesterase PaeY are secreted by this system (12, 33). The endo-pectate lyases are the major pectinolytic enzymes produced by E. chrysanthemi, and they play an important role in the maceration of plant tissues. These enzymes randomly cleave, by β-elimination, internal glycosidic linkages in pectic polymers, preferentially polygalacturonate or pectins with small numbers of methoxyl groups, and generate a series of oligogalacturonates with a 4,5-unsaturated residue at the nonreducing end.

Besides these extracellular enzymes, several cell-bound pectinases have been identified in E. chrysanthemi: the outer-membrane pectin methylesterase PemB (31), the periplasmic exopolygalacturonate lyase PelX and exo-α-d-polygalacturonosidase PehX (2, 8, 34), and the cytoplasmic oligogalacturonate lyase Ogl (3, 29). The two periplasmic exopectinases act on different extremities of the polymeric chain: PelX catalyzes the formation of unsaturated digalacturonates by attack from the reducing end, while PehX releases digalacturonates by attack from the nonreducing end (4, 34). The cytoplasmic oligogalacturonate lyase Ogl, like the pectate lyases, cleaves glycosidic linkages by β-elimination, but in contrast to these enzymes, it is only active on short oligogalacturonates (22).

Pectate lyases are classified into five different families according to their primary amino acid sequences (15, 36). The E. chrysanthemi isoenzymes are distributed among these five families. Family 1 contains several bacterial pectate lyases, fungal pectin lyases, and plant proteins (9, 10, 36). It includes the E. chrysanthemi isoenzymes PelA to PelE, which are further separated into two subfamilies, PelADE and PelBC (39). Family 3 includes PelB/Pel-3 of Erwinia carotovora, PelI of E. chrysanthemi (36), and the four Pel proteins of the phytopathogenic fungus Nectria haematococca (Fusarium solani) (7). The endo-pectate lyase PelL (1, 18) and the exopolygalacturonate lyase PelX of E. chrysanthemi are the only members of family 4 (2, 34). PelZ of E. chrysanthemi is the sole characterized component of the fifth family (26). Family 2 contains the periplasmic pectate lyases PelB of Erwinia carotovora (11) and PelY of Yersinia pseudotuberculosis (20). Despite its homology with family 2 pectate lyases, no pectinolytic activity was observed for the product of the E. chrysanthemi kdgC gene (5).

In this study, we showed that the kdgC gene of E. chrysanthemi 3937 is longer than previously supposed (5). The corresponding protein was overproduced, and we demonstrated that it encodes an exopolygalacturonate lyase. Hence, the kdgC gene was renamed pelW. The biochemical properties of PelW and its cellular localization were analyzed. To clarify the role of this cytoplasmic enzyme in pectin degradation, we compared its enzymatic properties with those of the other E. chrysanthemi cytoplasmic pectinase, Ogl.

MATERIALS AND METHODS

Strains, media, and growth conditions.

Bacterial strains and plasmids used in this study are listed in Table 1. Cells were grown in either Luria-Bertani (LB) or M63 medium (21). When required, the media were solidified with agar (15 g · liter⁻¹). E. chrysanthemi cells were usually incubated at 30°C, and Escherichia coli cells were generally incubated at 37°C. Pectate agar medium was used to detect pectinolytic activity (14). Carbon sources were added at 2 g · liter⁻¹ except for polygalacturonate (PGA) (grade II; Sigma), which was added at 4 g · liter⁻¹. When required, antibiotics were added at the following concentrations: kanamycin, 20 μg · ml⁻¹; ampicillin, 50 μg · ml⁻¹; and chloramphenicol, 20 μg · ml⁻¹.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant characteristics	Reference or source
Bacterial strains
E. chrysanthemi 3937 derivatives
A350	lacZ2	5
A1077	lacZ2 kdgR::Cm	Laboratory collection
A1671	lacZ2 pelW::MudI1734 Km	5
A3439	lacZ2 kdgR::Cm pelW::MudI1734 Km	This work
Escherichia coli
NM522	Δ(lac-proAB) Δ(mcrB-hsdSM)5 supE thi (F′ proAB lacIqlacZΔM15)	Laboratory collection
BL21(DE3)	E. coli B; F−dcm ompT hsdS gal λ(DE3); T7 polymerase gene under the control of the lacUV5 promoter	Stratagene
K-38	HfrC(λ) phoA4 pit-10 tonA22 ompF627 relA1	Laboratory collection
Plasmids
pBS Ap	pBluescript SK(+); Apr	Stratagene
pET-20b(+)	Carries the N-terminal pelB signal sequence; Apr	Novagen
pGP1.2	The T7 RNA polymerase gene under the control of the λPL promoter and the cI857 repressor; Kmr	38
pT7-6	T7Φ10; Apr	38
pNV9	pBR325 derivative carrying the 3.2-kb EcoRV-EcoRI fragment with pelW	5
pBS-KC	pBS derivative with the 2.9-kb EcoRV-ClaI fragment from pNV9; pelW+	This work
pT7-KC	pT7-6 derivative with the 3-kb EcoRV-EcoRI fragment from pNV9; pelW+	This work
pT7-KCsp	pET-20b(+) derivative with the 2.5-kb HpaI-HindIII ′pelW fragment under the control of the pelB signal sequence	This work
pOGL-10	pBS derivative carrying ogl	28
pT7-OGL	pT7-5 derivative with the 1.6-kb PinAI-PstI fragment from pOGL-10; ogl+	This work
pTaX	pT7-5 derivative with pelX; Apr	34
pT7-pelD	pT7-5 derivative with pelD; Apr	39

Protein production and cell fractionation.

The pelW gene was overexpressed by using the T7 promoter-T7 RNA polymerase system (38). The 2.9-kb EcoRV-ClaI fragment of pelW was inserted into the pT7-6 expression vector. The resulting plasmid, pT7-KC, was introduced into E. coli BL21(DE3). The BL21(DE3)/pT7-KC cells were grown at 30°C in LB medium supplemented with ampicillin (150 μg · ml⁻¹). At an optical density at 600 nm (OD₆₀₀) of 0.8 to 1, 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added, and the cells were grown for an additional 2 to 3 h. The cells were harvested by centrifugation for 10 min at 5,000 × g and 4°C and were subjected to osmotic shock for extraction of the overproduced protein (6).

For Ogl overproduction, the E. coli BL21(DE3)/pT7-OGL cells were cultivated as described above, except that the culture was grown for 12 to 14 h after IPTG addition. The cells were harvested by centrifugation and disrupted in a French press in 50 mM Tris-HCl (pH 8.0) with Complete protease inhibitor (Boehringer). The crude cell envelope fraction was eliminated by centrifugation at 100,000 × g for 1 h, and the supernatant was used directly for enzyme analysis.

For the E. chrysanthemi subcellular fractionation, the cells were grown in LB medium supplemented with galacturonic acid at 2 g · liter⁻¹. At an OD₆₀₀ of 1.2 to 1.5, spheroplasts were prepared as described by Witholt et al. (40).

Protein labelling.

Plasmid-encoded proteins were exclusively labelled with [³⁵S]cysteine-[³⁵S]methionine by using the T7 promoter-T7 polymerase system (38). For the cell fractionation experiments, cells were labelled for 10 min. Inhibition of the signal sequence processing was performed by adding 0.1 mM carbonylcyanide-m-chlorophenylhydrazone (CCCP) into an induced cell culture 2 min after addition of [³⁵S]cysteine-[³⁵S]methionine. The labelled amino acids were chased 3 min later, and cells were lysed in Laemmli sample buffer.

Analytical procedures.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (16a). Isoelectric focusing (IEF) was performed in a pH 3.5 to 10 gradient by using Pharmalytes. For differential detection of various pectate lyase activities after IEF, the gel was incubated in 50 mM Tris-HCl (pH 8.5) plus 5 g of PGA · liter⁻¹ with either 1 mM CaCl₂ (for extracellular pectate lyase detection) or 1 mM CoCl₂ (for PelW detection). After 10 to 60 min of incubation at 25°C, the gel was rinsed with water and stained with 0.05% ruthenium red. PelW activity is sensitive to the position of the sample application papers on the IEF gel; it must be placed in the area of neutral or slightly alkaline pH.

Thin-layer chromatography was used to identify the enzymatic degradation products of PGA and oligogalacturonates (18).

Enzyme assays.

Pectate lyase activity was determined by monitoring spectrophotometrically the formation of unsaturated products from substrate at 230 nm. Unless otherwise specified, the standard assay mixture consisted of 50 mM Tris-HCl (pH 8.5), 0.1 mM CaCl₂, and 0.5 g of PGA · liter⁻¹. The appearance of products was monitored at 37°C over a period of 1 min (at 6-s intervals). The molar extinction coefficient of unsaturated oligogalacturonates used was 5,200 (23). One unit of activity was defined as the amount of enzyme required to produce 1 μmol of unsaturated product per minute. To assay the PelW activity, 0.1 mM MnCl₂ was substituted for the CaCl₂ and 0.1 mM trigalacturonic acid (Sigma) was substituted for the PGA.

The alkaline phosphatase assay was performed with p-nitrophenyl phosphate as the substrate (19), and β-galactosidase activity was determined with o-nitrophenyl-β-d-galactoside as the substrate (21).

Recombinant DNA techniques.

Preparation of plasmid DNA, restriction digestions, ligations, DNA electrophoresis, and transformations were carried out as described by Sambrook et al. (30). Sequencing were performed by Genome Express SA (Grenoble, France).

Nucleotide sequence accession number.

The nucleotide sequence of the pelW (kdgC) gene has been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession no. X62073.

RESULTS

Determination of the nucleotide sequence of the kdgC gene.

The 2.3-kb EcoRV-PstI fragment containing the kdgC gene had been previously sequenced (5). However, no pectinolytic activity was found to be associated with the protein encoded by this fragment even after overproduction in different expression systems (data not shown). We decided to verify the sequence of this DNA region, mainly in the 5′ and 3′ ends of the potential open reading frame. This analysis revealed an error at the 3′ end of the open reading frame which extends beyond the PstI site. The corrected gene is 1,659 nucleotides long and encodes a 553-amino-acid protein with a deduced molecular mass of 64,082 Da and a calculated pI of 7.7. Subcloning a 2.9-kb EcoRV-ClaI fragment downstream of the plac promoter of pBluescript (pBS-KC) allowed us to detect a moderate pectate agar pitting activity around the E. coli NM522 colonies carrying this plasmid. Assay of pectinolytic activities revealed a weak pectate lyase activity in the corresponding cell lysates. Thus, this pectate lyase gene was renamed pelW.

The deduced amino acid sequence of PelW shows 35% identity with PelY of Y. pseudotuberculosis and 34% identity with PelB of E. carotovora (Fig. 1). The level of similarity decreases in the C-terminal parts of these proteins. However, the most important difference between PelW and these two proteins concerns their N-terminal ends. While PelY and PelB possess a typical signal peptide sequence, no signal sequence was identified within the PelW protein sequence.

FIG. 1.

Comparison of the amino acid sequences of the family 2 pectate lyases. The PelY sequence of Y. pseudotuberculosis was determined by Manulis et al. (20), and PelB of E. carotovora was described by Hinton et al. (11). Vertical lines indicate identical residues. The putative signal sequence cleavage sites of PelY and PelB are indicated by triangles.

PelW identification and determination of its cellular localization.

The PelW protein was overproduced in E. coli BL21(DE3)/pT7-KC cells (data not shown). A 65-kDa protein was detected by exclusive labelling of the plasmid-encoded proteins with [³⁵S]cysteine-[³⁵S]methionine (Fig. 2). Cell fractionation studies showed that this protein accumulated in the cytoplasm and that it was partially liberated by osmotic shock (Fig. 2).

FIG. 2.

Cellular localization of PelW in E. coli. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gels were autoradiographed. E. coli BL21(DE3)/pT7-KC (PelW), E. coli BL21(DE3)/pT7-6 (pT7-6), E. coli BL21(DE3)/pT7-KCsp (PelW-SP), and E. coli K38/pGP1.2/pT-pelD (PelD) cells were fractionated and then labelled with [³⁵S]cysteine-[³⁵S]methionine in the absence (−) or in the presence (+) of CCCP. W, whole-cell lysate; P, periplasmic fraction; C, osmotically shocked cell fraction. Precursor (p) and mature (m) forms of PelW-SP and PelD are indicated by arrowheads.

Since the two homologues of PelW, PelY and PelB, possess a signal peptide sequence and are periplasmic when expressed in E. coli (11, 20), we verified PelW localization in this host. No PelW was associated with the crude membrane fraction. Up to 30% of the total PelW enzymatic activity was released from E. coli cells by osmotic shock. However, the relative amount of osmotically released PelW depended on the strain used and on the protein production level and was significantly smaller than that of alkaline phosphatase (data not shown). This indicated that the partial extraction of PelW from the cells by osmotic shock was not due to a periplasmic localization of the protein but resulted from the partial leakage of overproduced PelW. Johnson and Hecht (13) showed that a nonspecific treatment of E. coli cells, e.g., repeated cycles of freezing and thawing, specifically released highly expressed recombinant protein from the bacterial cytoplasm without liberation of the bulk of the endogenous cytoplasmic E. coli proteins.

To exclude the possibility of the existence of a signal peptide for PelW, the protein was labelled and the cells were treated with CCCP, which stops signal peptide processing by dissipating the proton motive force (24). In contrast to PelD, which possesses a signal sequence, only one form of PelW was detected in the presence or in the absence of CCCP (Fig. 2). Moreover, PelW released by osmotic shock and PelW associated with the cells have the same molecular mass. Thus, as indicated by the sequence data, PelW does not possess a signal peptide and is located in the cytoplasm when produced in E. coli.

The differences observed among the N termini of PelW, PelB, and PelY led us to replace the 22 N-terminal amino acids of PelW with a classic signal peptide to construct a periplasmic derivative of PelW, PelW-SP. This chimeric protein was correctly maturated in E. coli cells and exported into the periplasm (Fig. 2 and data not shown). However, the resulting periplasmic PelW derivative was unstable, and no pectate lyase activity was found in association with this protein.

To determine the localization of PelW in E. chrysanthemi 3937, we used differential detection of PelW activity after IEF. This enzyme is activated in the presence of Co²⁺ cations, while the major E. chrysanthemi pectate lyases are inhibited under these conditions (39). Thus, to detect the PelW activity without interference from the major pectate lyases, we replaced Ca²⁺ with Co²⁺ for the detection of pectate lyase activities. A single band with an apparent pI of about 5.0 was detected in the extract of E. coli BL21(DE3)/pT7-KC cells overproducing PelW (Fig. 3). To increase PelW production in E. chrysanthemi, we used the kdgR mutant A1077, in which the pelW gene was shown to be derepressed (5). The band corresponding to PelW was detected in the cytoplasmic fraction of the A1077 strain but not in the periplasm or culture supernatant (Fig. 3). This band was absent from the pelW mutant A3439. Therefore, PelW is located in the cytoplasm of E. chrysanthemi 3937.

FIG. 3.

Cellular localization of PelW in E. chrysanthemi. IEF was followed by specific detection of pectate lyase activity in the presence of either CaCl₂ for 10 min (lanes 1 to 3) or CoCl₂ for 40 min (lanes 4 to 10). The osmotic-shock-released fraction of BL21(DE3)/pT7-KC (PelW⁺) (lane 7) was used as a control. Subcellular fractionation of E. chrysanthemi A1077 (kdgR) (lanes 1 to 6) and E. chrysanthemi A3439 (kdgR pelW) (lanes 8 to 10) was performed. The positions of the five major E. chrysanthemi pectate lyases (PelA to PelE), PelX, and PelW are indicated. S, culture supernatant; P, periplasm; C, spheroplast lysate.

Ogl production and assay.

Since PelW is a cytoplasmic pectate lyase, we decided to compare its properties with those of the other known cytoplasmic pectinase, the oligogalacturonate lyase Ogl. Overexpression of ogl in the E. coli recombinant strain BL21(DE3)/pT7-OGL yielded a high Ogl production level (about 0.4 mg · ml⁻¹ of the culture), and the enzyme constituted about 90% of the cell protein content (data not shown). Therefore, the soluble cell fraction from BL21(DE3)/pT7-OGL was directly used for the enzyme analysis.

Ogl activity is usually estimated by the periodate-thiobarbiturate assay (22). We proposed a direct UV detection at 230 nm for measurement of Ogl activity. We observed that the action of Ogl on digalacturonic acid led to an increase in absorbance at 230 nm. Such an increase in absorbance is observed during the formation of 4,5-unsaturated oligogalacturonates by pectate lyases. However, the UV-detected product appeared unstable, and several minutes after the start of the enzymatic reaction, the increase in OD₂₃₀ was followed by a successive decrease in absorbance. d-Galacturonic acid and 4-deoxy-l-threo-5-hexosulose uronic acid (DKI) have been identified as the Ogl products formed from digalacturonic acid (3, 22). Since these two products do not absorb at 230 nm, we proposed the following explanation for this phenomenon. The Ogl cleavage could give a 4,5-unsaturated pyranose monomer that, like 4,5-unsaturated digalacturonate, absorbs at 230 nm. The 4,5-unsaturated pyranose cycle could spontaneously produce a straight-chain 4,5-enolic form, which could then isomerize into the more stable 5-keto form (DKI). In spite of the relative instability of the first product formed by Ogl, it is possible to measure a linear increase in OD₂₃₀ during short assay periods (20 to 90 s). Thus, a direct UV detection at 230 nm was routinely used to measure Ogl activity.

Substrate specificity of PelW and Ogl.

To analyze the enzymatic properties of PelW, we used the fraction released by osmotic shock from E. coli BL 21(DE3)/pT7-KC. Despite the smaller amount of PelW in this fraction than in the total-cell lysate, the enzyme constituted about 20% of the total protein content of this fraction. The PelW enzymatic activity was first detected by using PGA as a substrate in the standard pectate lyase assay medium. Like the other E. chrysanthemi pectate lyases, PelW is active within a range of alkaline pH values, with an optimum at pH 8.5 in Tris-HCl buffer. In accordance with the cellular localization of this enzyme, it seemed unlikely that the polymer could be its natural substrate. Therefore, we analyzed the PelW activity on short oligogalacturonates, di-, tri-, and tetragalacturonate (G2, G3, and G4, respectively), which are more likely to be present in the E. chrysanthemi cytoplasm (Table 2). PelW did not cleave G2, while G3 and G4 were good substrates. The maximal activity was observed with G3. Determination of the K_m and V_max indicated that PelW exhibits the highest activity and affinity for G3 (Table 2). PGA was cleaved by this enzyme less effectively then G3 or G4. In comparison with the other E. chrysanthemi pectate lyases, PelW exhibits a low specific activity on PGA but a good affinity for this substrate. The higher activity observed with G3, compared with G4 or the polymer, suggested that the number of binding subsites for PelW was three, since the array of subsites is completely covered by G3. PelW shows almost the same initial reaction rate on PGA and on pectins with degrees of methylation up to 60% (data not shown). The enzyme retains about 50% of its maximal activity on 75% methylated pectin.

TABLE 2.

Effect of substrate length on PelW and Ogl activities

Substrate	Enzyme activitya ± SD
	PelW			Ogl
	Vob (U · mg−1)	Vmaxc (U · mg−1)	Kmc	Vod (U · mg−1)	Vmaxc (U · mg−1)	Kmc (μM)
G2	0			57 ± 3	80 ± 5	150 ± 30
G3	25 ± 2	30 ± 4	7 ± 2 μM	10 ± 2	20 ± 7	120 ± 50
G4	17 ± 2	23 ± 3	8 ± 2 μM	4 ± 1.5	NDe	ND
PGA	14 ± 0.4	6 ± 2	0.01 ± 0.004 g liter−1	0

^aNo enzymatic activity was detected on any of these substrates when E. coli extracts from vector-only-carrying strains were used. 

^bThe initial enzymatic rate of PelW was measured in 0.1 M Tris-HCl (pH 8.5) containing 0.1 mM MnCl₂ and either 0.5 g of PGA · liter⁻¹ or 50 μM oligogalacturonate (Gn, where n = 2 to 4). 

^cFor V_max and K_m determinations, PelW was incubated with PGA at concentrations of 0.005 to 0.025 g · liter⁻¹ and with oligogalacturonates at concentrations ranging from 5 to 40 μM, while Ogl was incubated with oligogalacturonates at concentrations ranging from 50 to 400 μM. 

^dThe initial enzymatic rate of Ogl was measured in 0.1 M Tris-HCl (pH 7) containing 0.1 mM MnCl₂ and either 0.5 g of PGA · liter⁻¹ or 400 μM oligogalacturonate. 

^eND, not determined because the activity of Ogl with these substrates was too low, which led to a high level of variability in the experimental data. 

To determine precisely the mode of action of PelW, the reaction products obtained from the action of this enzyme on various substrates were characterized by thin-layer chromatography (Fig. 4). The activity of PelW was compared with those of the products obtained after action of the E. chrysanthemi periplasmic exopolygalacturonate lyase PelX. With PGA as a substrate, PelW catalyzes the formation of only one product, unsaturated digalacturonate. The same product was liberated by the action of PelX. Thus, PelW is a pectate lyase with an exo-cleaving mode. The smallest substrates cleaved by PelW or PelX are the saturated and unsaturated trimers.

FIG. 4.

Identification of PelW and Ogl reaction products by thin-layer chromatography. The reaction mixtures contained 0.1 M Tris-HCl (pH 8), 0.1 mM MnCl₂, 5 U of enzyme ml⁻¹, and 1.5 mg of the following substrates · ml⁻¹: digalacturonic acid (G2), trigalacturonic acid (G3), a mixture of unsaturated di- and trigalacturonic acids (uG2, uG3), a mixture of unsaturated oligogalacturonic acids (uG3 to uGn), and polygalacturonic acid (PGA). Incubations were performed at 30°C for 3 h after addition of PelW, Ogl, or PelX or without enzyme (−). A 5-μl sample from each reaction was applied to a chromatogram sheet. The positions of the individual compounds are indicated by arrowheads. G1 and DKI are at the same position, but the staining method was not sensitive enough to detect the amounts of DKI applied to the chromatogram; 10 μg of DKI or G1 was applied to the lanes indicated by asterisks.

The enzymatic properties of Ogl were analyzed by using the soluble cell fraction of E. coli BL21(DE3)/pT7-OGL. The optimum pH of Ogl activity determined in 50 mM Tris-HCl buffer was between 7.3 and 7.7. Analysis of the action of Ogl on oligogalacturonates of different lengths (G2, G3, and G4) showed that the dimer was the best substrate for the enzyme (Table 2). Ogl cleaves both saturated and unsaturated dimers (Fig. 4). A large decrease in Ogl activity was observed for G3, while G4 was a very poor substrate (Table 2). Similarly, unsaturated oligogalacturonates larger than unsaturated G3 were not effectively cleaved by Ogl (Fig. 4). No enzymatic activity on polymeric substrates, such as PGA or pectins, could be detected.

Cation requirements of PelW and Ogl.

The characterized E. chrysanthemi pectate lyases have an absolute requirement for Ca²⁺. Addition of EDTA totally inhibits PelW, demonstrating its absolute cation requirement (Fig. 5A). PelW is weakly affected by the addition of Ca²⁺ but is strongly activated by Co²⁺, Mn²⁺, and Ni²⁺, with a maximum at concentrations of 0.1 to 1 mM (Fig. 5C and data not shown). Co²⁺ is the best cofactor for the enzyme. The effect of these cations is not additive, indicating that they have the same target on PelW and/or its substrate. It should be noted that the cation content of the commercially available G3 and PGA is high enough to give about 50% of the maximal PelW activity without addition of any cation (Fig. 5A and C).

FIG. 5.

Cation requirements of PelW and Ogl. The cation requirements of PelW (A) and Ogl (B) were tested by using 0.1 mM concentrations of their corresponding chloride forms, 25 μM EDTA, and 0.1 mM G3 in 0.1 M Tris-HCl (pH 8.5) as a substrate for PelW and 0.67 mM G2 in 0.1 M Tris-HCl (pH 7.0) as a substrate for Ogl. The dotted lines show the levels of enzyme activities in the absence of added EDTA or cations. −, no cation added. (C) The influence of Mn²⁺ concentration on PelW activity was tested in 0.1 M Tris-HCl (pH 8.5)–0.1 mM G3, using various concentrations of EDTA or MnCl₂. (D) The thermostability of PelW was monitored at 45°C after various incubation times in 0.1 M Tris-HCl (pH 8.0) containing 0.4 mM EDTA, either alone or with a 1 mM concentration of CaCl₂, CoCl₂, or MnCl₂. The residual activity is given as a percentage of the initial activity.

Binding of Ca²⁺ to the extracellular pectate lyases increases their stability (32, 39). We analyzed whether the stability of PelW is affected by the presence of cations. The stability of the protein was studied by measuring its thermal inactivation at 45°C. In the presence of EDTA, PelW lost 90% of its initial activity in 2 min (Fig. 5D). While addition of Ca²⁺ to PelW did not modify its stability, the presence of Mn²⁺ or Co²⁺ led to an essential increase in the enzyme’s stability, since the half-life of PelW increased to 4 or 15 min, respectively (Fig. 5D).

We tested Ogl’s cation requirement by using G2 as a substrate. Addition of 25 μM EDTA completely inhibited the Ogl activity (Fig. 5B). Among the divalent cations tested, Mn²⁺, Co²⁺, Fe²⁺, and Ni²⁺ strongly activated the enzyme. Mn²⁺ was the best cofactor for Ogl (Fig. 5B), with an optimal concentration of 0.1 mM (data not shown). Ca²⁺ had no effect on the enzyme activity.

DISCUSSION

This paper reports the characterization of PelW, the cytoplasmic exopolygalacturonate lyase of E. chrysanthemi 3937. It has been previously shown that the product of the kdgC gene is homologous to family 2 pectate lyases (5). However, an error in the gene sequence resulted in the failure of the authors to purify an active protein. We renamed this gene pelW after demonstrating that it encodes an exo-cleaving pectate lyase. It is not known whether the two other members of family 2, PelY of Y. pseudotuberculosis (20) and PelB of E. carotovora (11), are endo- or exo-cleaving enzymes. Their homology with PelW is not sufficient to infer a similar mode of substrate recognition. Indeed, pectate lyase family 3 includes both an exo- and an endo-cleaving enzyme, PelX and PelL, respectively (18, 34). Moreover, these enzymes are not situated in the same cellular compartment; PelL is extracellular, while PelX is located in the periplasm. Similarly, the cellular localization of the family 2 pectate lyases can differ. Both PelY and PelB possess a typical signal sequence and are periplasmic when expressed in E. coli (11, 20). We demonstrated that PelW has no signal sequence and is located in the cytoplasm. Thus, in addition to a set of at least eight extracellular endo-pectate lyases (36), E. chrysanthemi produces two intracellular exopolygalacturonate lyases, the periplasmic PelX and the cytoplasmic PelW. It is remarkable that these 10 pectate lyases are distributed among the five defined pectate lyase families, implying independent genetic acquisitions.

A cell-bound exo-pectate lyase activity was first reported in E. chrysanthemi CUCPB1237 (3). The pelX gene was then cloned from E. chrysanthemi EC16 (2) and E. chrysanthemi 3937 (34). Evidence that PelW, like PelX, is an exo-cleaving lyase arises from the fact that its action on a polymeric substrate generates a single product, unsaturated digalacturonate (Fig. 4). In contrast, an endo-cleaving enzyme would catalyze the formation of multiple products of various sizes (27). Despite the fact that the two E. chrysanthemi exopolygalacturonate lyases form the same product from the polymeric substrate PGA and show relatively good activity on this substrate, they exhibit some differences in activity on various oligomers. PelX shows a high level of activity on oligomers from G4 to G7, with a maximal activity on G4. G3 is a poor substrate for PelX (34). On the contrary, G3 is the best substrate of PelW, while this enzyme shows a lower level of activity on G4. The cellular localization of these two exopolygalacturonate lyases seems to be complementary. PelX could cleave oligomers when they pass through the periplasm, while PelW could cleave oligomers entering the cytoplasm. Thus, the two exopolygalacturonate lyases could ensure successive depolymerization of pectic substances, and their substrate preferences are linked to their cellular compartments. The data obtained with oligomers suggest that the number of subsites of PelW is only three (−1 to +2); PelW seems to recognize only three residues at the reducing end of the polysaccharide. In contrast to the other pectate lyases, PelW is highly tolerant of the methylation of the substrate. It shows almost the same activity on PGA as it does on pectins with a degree of methylation up to 60%. Two E. chrysanthemi pectin methylesterases, the extracellular PemA and the outer-membrane-located PemB, are responsible for the demethylation of pectic substances in the extracellular medium and in the periplasm, respectively (17, 31). Nevertheless, the cytoplasmic lyase PelW might be able to cleave methylated oligomers escaping from the demethylation by these pectin methylesterases.

To better understand the role of the cytoplasmic pectinases in the final steps of pectin depolymerization in the E. chrysanthemi cytoplasm, we compared the properties of PelW with those of the oligogalacturonate lyase Ogl, which was the only known Erwinia cytoplasmic pectinase (3, 22). Ogl from E. carotovora was previously purified, and its catalytic properties were analyzed (22). Although the E. chrysanthemi ogl gene was cloned and sequenced (28, 29), enzymatic properties of the corresponding protein have never been studied. Nevertheless, this enzyme seems to play a key role in the pectin catabolic pathway, since E. chrysanthemi ogl mutants are unable to grow on PGA or digalacturonates as a carbon source (3, 29). This enzyme links the extracellular and intracellular steps of the pectin catabolic pathway because it cleaves the dimers produced by the combined action of all the other pectinases. However, the role of PelW in pectin catabolism is not negligible, since the E. chrysanthemi pelW (kdgC) mutant shows reduced growth on PGA (5). The role of the two enzymes PelW and Ogl is clearer when their substrate specificities are compared. Oligomers longer than dimers are very poor substrates for Ogl, while the trimer is the best substrate for PelW, which retains a good activity on the tetramer. Thus, these two enzymes seem to act in sequence during oligogalacturonate depolymerization in the cytoplasm. The substrate specificity of PelW implies that trimers, and probably tetramers, enter the E. chrysanthemi cytoplasm. The transport of pectic oligomers into the bacterial cells is the least-well-known step of pectin catabolism. Numerous mutants of E. chrysanthemi were isolated for their inability to grow on medium with PGA as the carbon source. None of them appeared to be affected with regard to transport of pectic oligomers. This result led us to theorize that several systems could be used for oligogalacturonate uptake.

The extracellular E. chrysanthemi endo-pectate lyases demonstrate an absolute Ca²⁺ requirement for their enzymatic activity (18, 25, 36, 39). These enzymes either are not affected or are inhibited in the presence of other divalent cations, with the exception of PelZ, which uses Mn²⁺ as a cofactor more efficiently than it uses Ca²⁺ (25, 39). The periplasmic exopolygalacturonate lyase PelX also requires cations for its activity and is weakly activated by Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, or Cu²⁺ (34). It has been previously reported that the E. carotovora Ogl does not require calcium ions and is not inactivated by EDTA (22). Our results show that the activities of the two E. chrysanthemi cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. However, Ca²⁺ is unable to restore their activity, while several other metal cations of similar size, such as Co²⁺, Mn²⁺, and Ni²⁺, can activate both PelW and Ogl with different efficiencies (Fig. 5A and B). The PelW thermoinactivation data indicate a direct binding of cation to the protein, resulting in the stabilization of the protein structure (Fig. 5D). It is remarkable that both cytoplasmic lyases are activated by almost the same spectrum of cations, which is very different from the cation requirement of the extracellular pectate lyases. Ca²⁺ binds to the extracellular pectate lyases and also interacts with the carboxyl groups of their substrate, PGA. These cations have been shown to be directly involved in catalysis (16). It is possible that the cation dependence of PelW and Ogl is related to specific features of the catalytic reactions carried out by these enzymes. However, the substrate specificities and the reaction products of PelW and Ogl are quite different. Moreover, except for the cation requirement, the enzymatic properties of PelW do not essentially differ from those of PelX or of the endo-pectate lyases. Another explanation for the similar cation requirement spectra of PelW and Ogl could be related to their cytoplasmic localization. The bacterial cells maintain an intracellular calcium level below that of the growth medium. For example, in E. coli, the intracellular calcium concentration is 0.1 μM, regardless of its extracellular concentration (37). On the other hand, several specific transport systems ensure the intracellular uptake of numerous other divalent metal cations (37). Thus, the cation content of the bacterial cytoplasm could probably constrain the bacteria to use other divalent cations in the place of Ca²⁺ for similar catalytic processes.