Mutagenesis of Endopolygalacturonase from<i>Fusarium moniliforme:</i>Histidine Residue 234 Is Critical for Enzymatic and Macerating Activities and Not for Binding to Polygalacturonase-lnhibiting Protein (PGIP)

Caprari, Claudio; Mattei, Benedetta; Basile, Maria Luisa; Salvi, G.; Crescenzi, V.; Lorenzo, Giulia De; Cervone, Felice

doi:10.1094/mpmi-9-0617

Cited by 68 publications

(34 citation statements)

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“…The x-ray structure of FmPG can help us to understand how the enzyme forms a complex with P. vulgaris PGIP-2 and why the formation of the complex leads to the inhibition of the enzyme activity. We have established that, like the residues His-234, Ser-237, and Ser-240 reported in a previous paper (19), also the catalytic residues Asp-191, Asp-212, and Asp-213 of FmPG do not form contacts with PGIP-2. Instead, three other residues are important for the formation of the complex: two of them (Lys-269 and Arg-267) are putatively involved in substrate binding whereas to the third one (His-188), located at the opposite side of the active site cleft, has not been ascribed any defined role in catalysis.…”

Section: Discussionsupporting

confidence: 83%

“…FmPG and mutant FmPGs expressed in yeast were prepared and purified as previously described (19). Protein concentration was determined by the method of Bradford (20).…”

Section: Methodsmentioning

confidence: 99%

“…FmPG activity was determined by reducing endgroup analysis as already described (19). One activity unit (RGU, reducing groups unit) was defined as the amount of the enzyme producing 1 mol of reducing groups per min at 30°C with 0.5% (wt͞vol) polygalacturonic acid as substrate.…”

Section: Methodsmentioning

confidence: 99%

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Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

Federici

Caprari

Mattei

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

135

129

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To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 Å resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding.

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Section: Discussionsupporting

confidence: 83%

“…FmPG and mutant FmPGs expressed in yeast were prepared and purified as previously described (19). Protein concentration was determined by the method of Bradford (20).…”

Section: Methodsmentioning

confidence: 99%

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Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

Federici

Caprari

Mattei

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

135

129

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“…Cultures were incubated in a rotary shaker at 180 rpm and 21ЊC for 12 days, and culture filtrates were used for PG activity assay. PG II from Aspergillus niger was prepared as described (Armand et al, 2000), and PG of Fusarium moniliforme also was prepared as described (Caprari et al, 1996). PG activity was measured using a modified agarose diffusion assay (Taylor and Secor, 1988).…”

Section: Preparation and Assay Of Fungal Pgsmentioning

confidence: 99%

Tandemly Duplicated Arabidopsis Genes That Encode Polygalacturonase-Inhibiting Proteins Are Regulated Coordinately by Different Signal Transduction Pathways in Response to Fungal Infection

et al. 2002

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Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1 , whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.

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“…Cultures were incubated in a rotary shaker at 180 rpm and 21°C for 12 d, and filtrates were used for the PG activity assay. PGII of A. niger was prepared as described by Cervone et al (1987), and PG of Fusarium moniliforme expressed in Saccharomyces cerevisiae was prepared as described previously (Caprari et al, 1996). Lygus rugulipennis Poppius and Adelphocoris lineolatus (Goeze) were field collected from alfalfa (Medicago sativa) and laboratory reared on fresh green beans and sunflower kernels (25 6 2°C, 70% 6 10% relative humidity, 14-hlight:10-h-dark photoperiod).…”

Section: Preparation and Assay Of Polygalacturonases And Pgipsmentioning

confidence: 99%

Characterization of the Complex Locus of Bean Encoding Polygalacturonase-Inhibiting Proteins Reveals Subfunctionalization for Defense against Fungi and Insects

et al. 2004

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Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that\ud belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean\ud (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their\ud similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both\ud partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited\ud PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed\ud variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue\ud previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93\ud PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse\ud transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding,\ud and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are\ud important for the adaptation of plants to pathogenic fungi and phytophagous insects

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Mutagenesis of Endopolygalacturonase fromFusarium moniliforme:Histidine Residue 234 Is Critical for Enzymatic and Macerating Activities and Not for Binding to Polygalacturonase-lnhibiting Protein (PGIP)

Cited by 68 publications

References 0 publications

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

Tandemly Duplicated Arabidopsis Genes That Encode Polygalacturonase-Inhibiting Proteins Are Regulated Coordinately by Different Signal Transduction Pathways in Response to Fungal Infection

Characterization of the Complex Locus of Bean Encoding Polygalacturonase-Inhibiting Proteins Reveals Subfunctionalization for Defense against Fungi and Insects

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