Oligogalacturonide trans-eliminase of Erwinia carotovora

Moran, Francis; Nasuno, Seiichi; Starr, Mortimer P.

doi:10.1016/0003-9861(68)90508-0

Cited by 50 publications

(26 citation statements)

References 18 publications

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“…Evolution of the OGL Family-EcOGL was first discovered in 1968 (originally referred to as oligogalacturonide transeliminase (46)) and known to be found in diverse species of Enterobacteriaceae, including Y. enterocolitica, for over a decade (8); however, a detailed analysis of the relatedness of orthologs and its classification as a novel polysaccharide lyase family in the CAZy data base has been lacking. Therefore, to provide insight into the functional relatedness of the family, we did a BLAST search using the YeOGL as a query sequence.…”

Section: Resultsmentioning

confidence: 99%

The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination

Abbott

Gilbert

Boraston

2010

Journal of Biological Chemistry

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Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a ␤-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å . The model contains a Mn 2؉ atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the ␣-proton in the ؊1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases.

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Section: Resultsmentioning

confidence: 99%

The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination

Abbott

Gilbert

Boraston

2010

Journal of Biological Chemistry

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“…Total specific activity, expressed as AASS0 mg-' min-' ml-' and AA436 mg-' min-' ml-', for cellulase and protease respectively, was determined. Liquid assays of pectate lyase activity were performed on toluenized cell extracts as described previously (Moran et al, 1968 RexZ and KdgR control of exoenzyme genes was performed by the chain-termination method (Sanger et al, 1977) on random sonicated templates cloned into the M13 vector mp18 or mp19. PCR (using Tag DNA polymerase, NEB; annealing temperature 43 "C) was used to amplify the E. carotovora subsp.…”

Section: Methodsmentioning

confidence: 99%

Erwinia carotovora has two KdgR-like proteins belonging to the IcIR family of transcriptional regulators: identification and characterization of the RexZ activator and the KdgR repressor of pathogenesis

et al. 1999

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A novel Erwinia carotovora subsp. carotovora mutant designated RexZ, begulator of Eoenzymes) showed reduced production of the degradative exoenzymes. The mxZ gene product shows similarity to the KdgR regulatory protein from fmvinia chrysanthemi, described as the major repressor of the pectin catabolism pathway genes in the latter species. In vitro DNA-protein interaction experiments demonstrated that the synthesis of the Rex2 protein is controlled by the cAMP-CRP (CAMP-receptor protein) complex. Western blot analysis also revealed the presence of a second KdgR homologue (distinct from RexZ) which, like RexZ, was present in all species of the genus ErWinia tested. The corresponding KdgR proteins from both Em carotovora subsp. carotovora and E carotovora subsp. afroseptica share a high level of sequence identity with the KdgR homologues from E. chrysantherni and Escherichia coli.Although the E. carotovora subsp. carotovora rexZ regulatory region displayed specific interactions with both the purified E. chrysanthemi KdgR repressor and the partially purified E. carotovora subsp. carotovora KdgR, in vivo quantification revealed that the cetlular levet of Rex2 protein was unaffected by the presence of pectic compounds. This study shows that the complex regulatory network governing virulence in the erwinias involves two total ty distinct, but highly conserved, members of the IclR class of DNA binding proteins: RexZ and KdgR. INTRODUCTIONThe phytopathogenicity of several Erwinia species is correlated with the ability to produce and secrete plant cell wall degrading enzymes such as pectinases, cellulases (Cel) and proteases (Prt) (Collmer & Keen, 1986;Barras et al., 1994). The crucial role of these enzymes, particularly the pectate lyases (Pel), in the Abbreviations: CRP, cyclic AMP receptor protein; KDG, 2-keto-3-deoxygluconate; OHHL, N-(3-oxohexanoyt)-~-homoserine lactone; PGA, polygalacturonate.The GenBank accession numbers for the sequences reported in this paper are given in the text.virulence of soft-rot Erwinia has been confirmed by the isolation of mutants exhibiting reduced virulence that are defective for production or secretion of these enzymes (Boccara et al., 1988;Hinton et al., 1989; Pirhonen st al., 1991). Exoenzyme production by softrot Erwinia species responds to several environmental conditions (Hugouvieux-Cotte-Pattat et af., 1996) ; presence of pectin-degradative products or plant extract, anaerobiosis, temperature, nitrogen starvation, osmolarity, catabolite repression, iron availability and growth phase.In Erwinia chrysanthemi 3937, attention has been focused on the pectin degradation pathway, and the different steps of this metabolic pathway have been characterized. The degradation of the pectic compounds is initiated by extracellular pectinases, including two pectin methylesterases (encoded by pernA and pemB) (Laurent et al., 1993;Shevchik et al., 1996), five major isoenzymes of pectate lyases (encoded by pelA, pel& pelC, pelD and pelE) and a set of secondary pectate lyases which generate u...

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“…Each experiment was repeated at least three times. Pectate lyase activity was determined by monitoring spectrophotometrically at 230 nm the formation of unsaturated products from PGA (0.05%) in 0.1 M Tris-HCl (pH 8.5) buffer containing 0.1 mM CaCl 2 (24). One unit of activity is defined as the amount of enzyme required to produce 1 mol of unsaturated product per min.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the pecT control region from Erwinia chrysanthemi 3937

Castillo

Reverchon

1997

J Bacteriol

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Erwinia chrysanthemi synthesizes and secretes pectate lyases that attack components of the plant cell wall and, therefore, play a major role in the pathogenesis of soft rot disease. We isolated a new mutant (designated pec-1), by Tn5 mutagenesis, that displays weak pectate lyase production and decreased motility and mucoidicity. Maceration and pathogenicity tests done on different plant organs showed that the pec-1 strain displays a reduced virulence compared to that of the parental strain. The Tn5 insertion was localized between the pelL and the out loci and defines a new regulatory region. Sequencing of the pec-1::Tn5 insertion revealed that pec-1 is tightly linked to the pecT regulatory gene that also controls pectate lyase synthesis. Moreover, the pecT mutation is dominant over the pec-1 mutation, suggesting that these two loci are involved in the same regulatory network. We demonstrated, by Northern blot analysis, that the pec-1::Tn5 insertion provokes derepression of pecT transcription and defines a cis-acting element. Introduction of the pecT gene in trans of a pecT::uidA fusion induced a decrease of pecT::uidA transcription, indicating a negative autoregulation. Band shift experiments confirmed that the PecT repressor specifically interacts with the pecT regulatory region. We also demonstrated that the PecT protein interacts with the regulatory region of the pelD gene encoding a pectate lyase. Therefore, the abolition of the pecT autoregulation in the pec-1 mutant provokes an overproduction of the PecT repressor that is responsible for the decrease of pectate lyase synthesis. Mutagenesis of the pecT regulatory region revealed the presence of two sites in which insertions reproduced the pec-1 phenotype. This result suggests that pecT autoregulation requires the presence of two functional operator sites. From this study, we propose that the PecT repressor binds to these two sites, generating a loop that blocks pecT transcription.

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Oligogalacturonide trans-eliminase of Erwinia carotovora

Cited by 50 publications

References 18 publications

The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination

The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination

Erwinia carotovora has two KdgR-like proteins belonging to the IcIR family of transcriptional regulators: identification and characterization of the RexZ activator and the KdgR repressor of pathogenesis

Characterization of the pecT control region from Erwinia chrysanthemi 3937

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