NORMAL GENOMIC

• Di Matteo A et al.
• Structural basis for the interaction between pectin methylesterase and aspecific inhibitor protein.
• Plant Cell. 2005; 17: 849-58
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• Pectin, one of the main components of the plant cell wall, is secreted ina highly methyl-esterified form and subsequently deesterified in muro bypectin methylesterases (PMEs). In many developmental processes, PMEs areregulated by either differential expression or posttranslational controlby protein inhibitors (PMEIs). PMEIs are typically active against plantPMEs and ineffective against microbial enzymes. Here, we describe thethree-dimensional structure of the complex between the most abundant PMEisoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi(Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into aright-handed parallel beta-helical structure typical of pectic enzymes.The inhibitor is almost all helical, with four long alpha-helices alignedin an antiparallel manner in a classical up-and-down four-helical bundle.The two proteins form a stoichiometric 1:1 complex in which the inhibitorcovers the shallow cleft of the enzyme where the putative active site islocated. The four-helix bundle of the inhibitor packs roughlyperpendicular to the main axis of the parallel beta-helix of PME, andthree helices of the bundle interact with the enzyme. The interactioninterface displays a polar character, typical of nonobligate complexesformed by soluble proteins. The structure of the complex gives an insightinto the specificity of the inhibitor toward plant PMEs and the mechanismof regulation of these enzymes.

• Raiola A et al.
• Two Arabidopsis thaliana genes encode functional pectin methylesteraseinhibitors.
• FEBS Lett. 2004; 557: 199-203
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• We have identified, expressed and characterized two genes from Arabidopsisthaliana (AtPMEI-1 and AtPMEI-2) encoding functional inhibitors of pectinmethylesterases. AtPMEI-1 and AtPMEI-2 are cell wall proteins sharing manyfeatures with the only pectin methylesterase inhibitor (PMEI)characterized so far from kiwi fruit. Both Arabidopsis proteins interactwith and inhibit plant-derived pectin methylesterases (PMEs) but notmicrobial enzymes. The occurrence of functional PMEIs in Arabidopsisindicates that a mechanism of controlling pectin esterification byinhibition of endogenous PMEs is present in different plant species.

• Hothorn M, Wolf S, Aloy P, Greiner S, Scheffzek K
• Structural insights into the target specificity of plant invertase andpectin methylesterase inhibitory proteins.
• Plant Cell. 2004; 16: 3437-47
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• Pectin methylesterase (PME) and invertase are key enzymes in plantcarbohydrate metabolism. Inhibitors of both enzymes constitute a sequencefamily of extracellular proteins. Members of this family are selectivelytargeted toward either PME or invertase. In a comparative structuralapproach we have studied how this target specificity is implemented onhomologous sequences. By extending crystallographic work on the invertaseinhibitor Nt-CIF to a pectin methylesterase inhibitor (PMEI) fromArabidopsis thaliana, we show an alpha-helical hairpin motif to be anindependent and mobile structural entity in PMEI. Removal of this hairpinfully inactivates the inhibitor. A chimera composed of the alpha-hairpinof PMEI and the four-helix bundle of Nt-CIF is still active against PME.By contrast, combining the corresponding segment of Nt-CIF with thefour-helix bundle of PMEI renders the protein inactive toward either PMEor invertase. Our experiments provide insight in how these homologousinhibitors can make differential use of similar structural modules toachieve distinct functions. Integrating our results with previousfindings, we present a model for the PME-PMEI complex with importantimplications.

• Irifune K, Nishida T, Egawa H, Nagatani A
• Pectin methylesterase inhibitor cDNA from kiwi fruit.
• Plant Cell Rep. 2004; 23: 333-8
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• We have newly isolated one partial pectin methylesterase inhibitor (PMEI)and two full-length cDNA clones from a kiwi fruit cDNA library. The twofull-length cDNA clones, Adpmei-1 and Adpmei-2, had an open reading frameof 185 amino acids, including a predicted signal peptide sequencenecessary for localization in the cell-wall space. As the deduced aminoacid sequence of the cloned fragment was almost same as the sequence ofthe previously purified PMEI protein (Camardella et al., Eur J Biochem267:4561-4565), the clones were considered to be cDNAs encoding PMEIprotein. Southern blot analysis indicated a low-copy number of the PMEIgenes. Transgenic analysis of asparagus calli expressing a kiwi fruit PMEIgene driven by the CaMV 35S promoter demonstrated in vivo inhibitioneffects of PMEI on the endogenous pectin methylesterase (PME) activity.The relative expression levels of the PMEI genes in kiwi fruit, analyzedby competitive PCR, increased with the progression of fruit maturation.Given that PME activity also showed its highest level at the fully ripenedstage of maturation, the increase in PMEI expression may not indicatedirect inhibitory effects on the PME activity and fruit maturationprocess.

• Ly-Nguyen B, Van Loey AM, Smout C, Verlent I, Duvetter T, Hendrickx ME
• Effect of intrinsic and extrinsic factors on the interaction of plantpectin methylesterase and its proteinaceous inhibitor from kiwi fruit.
• J Agric Food Chem. 2004; 52: 8144-50
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• A proteinaceous pectin methylesterase inhibitor (PMEI) was isolated fromkiwi fruit (Actinidia chinensiscv. Hayward) and purified by affinitychromatography on a cyanogen bromide (CNBr) Sepharose 4B-orange PMEcolumn. The optimal pH of banana PME activity was 7.0, whereas that forcarrot and strawberry PME activity was 9.0. The optimal pH for the bindingbetween kiwi fruit PMEI and these PMEs was 7.0. The kiwi fruit PMEI has adifferent affinity for PME depending on the plant source. The inhibitionkinetics of kiwi fruit PMEI to banana and strawberry PME followed anoncompetitive type, whereas that to carrot PME followed a competitivetype. The kiwi fruit PMEI was mixed with banana, carrot, and strawberryPME to obtain PMEI-PME complexes, which were then subjected to thermal(40-80 degrees C, atmospheric pressure) or high-pressure (10 degrees C,100-600 MPa) treatment. Experimental data showed that the PMEI-PMEcomplexes were easily dissociated by both thermal and high-pressuretreatments.

• Ciardiello MA et al.
• Pectin methylesterase from kiwi and kaki fruits: purification,characterization, and role of pH in the enzyme regulation and interactionwith the kiwi proteinaceous inhibitor.
• J Agric Food Chem. 2004; 52: 7700-3
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• Pectin methylesterase was purified from kiwi (Actinidia chinensis) andkaki fruit (Diospyros kaki). The pH values of the fruit homogenates were3.5 and 6.2, respectively. The kiwi enzyme is localized in the cell walland has a neutral-alkaline pI, whereas the kaki enzyme is localized in thesoluble fraction and has a neutral-acidic pI. The molecular weights of thekiwi and kaki enzymes were 50 and 37 kDa, respectively. The two enzymesshowed a similar salt and pH dependence of activity, and a different pHdependence of the inhibition by the kiwi proteinaceous inhibitor.

• Scognamiglio MA, Ciardiello MA, Tamburrini M, Carratore V, Rausch T, Camardella L
• The plant invertase inhibitor shares structural properties and disulfidebridges arrangement with the pectin methylesterase inhibitor.
• J Protein Chem. 2003; 22: 363-9
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• Attempts to purify the inhibitor of pectin methylesterase (PMEI) from thesoluble extract of ripe apricot (Prunus armeniaca) fruit led to isolationof a protein (Pa-INH) similar to PMEI, but having invertase inhibitoryactivity against vacuolar invertase from tomato. The molecular charge, thenative and SDS-PAGE molecular weights were similar to those of PMEI.Partial amino acid sequence indicated a high level of identity withinvertase inhibitors and a significant identity with PMEI. Circulardichroism analysis showed a mainly alpha-helix secondary structure forboth the inhibitors and a higher thermostability of Pa-INH. Four Cysresidues forming disulfide bridges in PMEI were conserved in Pa-INH.Similarly to PMEI, these residues were linked by disulfide bridges (firstto second and third to fourth). The free Cys139 of PMEI is substituted byAla in Pa-INH. The results reported in this study suggest a commonstructural arrangement of the two inhibitors.

• Balestrieri C, Castaldo D, Giovane A, Quagliuolo L, Servillo L
• A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidiachinensis).
• Eur J Biochem. 1990; 193: 183-7
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• The finding of a powerful inhibitor of pectin methylesterase in ripe kiwifruit is reported. The inhibitor was revealed to be a glycoprotein. It waspurified to homogeneity and found to have a molecular mass of about 28kDa, as estimated by gel filtration chromatography, SDS/PAGE andanalytical ultracentrifugation. The sugar portion is composed ofgalactose, arabinose and rhamnose, the latter being much less represented.The amino acid composition showed a very high content of acidic residuescompared to basic ones, which is the reason for the very low isoelectricpoint of the protein (less than 3.5). The kind of inhibition on kiwipectin methylesterase was found to be competitive with an apparent Ki of0.22 microM, using citrus pectin as a substrate. Moreover, the inhibitoris effective in inhibiting pectin methylesterase in the pH range 3.5-7.5.Kiwi inhibitor appears to be specific for pectin methylesterase, inasmuchas it was found to be ineffective against other polysaccharide-degradingenzymes, such as polygalacturonase and amylase. Conversely, it appears tobe completely aspecific as far as the pectin methylesterase source isconcerned. In fact, it was found to inhibit this enzyme effectively fromall the sources we assayed, i.e. orange, tomato, apple, banana, potato.