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Comparative Study
. 1999 Jul;181(13):3912-9.
doi: 10.1128/JB.181.13.3912-3919.1999.

The exopolygalacturonate lyase PelW and the oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin catabolism in Erwinia chrysanthemi 3937

V E Shevchik  1 , G Condemine, J Robert-Baudouy, N Hugouvieux-Cotte-Pattat
Affiliations
Comparative Study

The exopolygalacturonate lyase PelW and the oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin catabolism in Erwinia chrysanthemi 3937

V E Shevchik et al. J Bacteriol. 1999 Jul.

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.

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Figures

FIG. 1
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.
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.
FIG. 3
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.
FIG. 4
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.
FIG. 5
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.
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