PelN is a new pectate lyase of Dickeya dadantii with unusual characteristics.

The plant-pathogenic bacterium Dickeya dadantii produces several pectinolytic enzymes that play a major role in the soft-rot disease. Eight characterized endopectate lyases are secreted in the extracellular medium by the type II secretion system, Out. They cleave internal glycosidic bonds of pectin, leading to plant tissue maceration. The D. dadantii pectate lyases belong to different families, namely, PL1, PL2, PL3, and PL9. Analysis of the D. dadantii 3937 genome revealed a gene encoding a new protein of the PL9 family, which already includes the secreted endopectate lyase PelL and the periplasmic exopectate lyase PelX. We demonstrated that PelN is an additional extracellular protein secreted by the Out system. However, PelN has some unusual characteristics. Although most pectate lyases require a very alkaline pH and Ca²âº for their activity, the PelN activity is optimal at pH 7.4 and in the presence of Fe²âº as a cofactor. PelN is only weakly affected by the degree of pectin methyl esterification. The PelN structural model, constructed on the basis of the PelL structure, suggests that the PelL global topology and its catalytic amino acids are conserved in PelN. Notable differences concern the presence of additional loops at the PelN surface, and the replacement of PelL charged residues, involved in substrate binding, by aromatic residues in PelN. The pelN expression is affected by different environmental conditions, such as pH, osmolarity, and temperature. It is controlled by the repressors KdgR and PecS and by the activator GacA, three regulators of D. dadantii pectinase genes. Since a pelN mutant had reduced virulence on chicory leaves, the PelN enzyme plays a role in plant infection, despite its low specific activity and its unusual cofactor requirement.

Molecular basis of the activity of the phytopathogen pectin methylesterase.

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel beta-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.

PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E. chrysanthemi. These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.

PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases.

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-alpha-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.