Tomato pectin methylesterase: Modeling, fluorescence, and inhibitor interaction studies—comparison with the bacterial (Erwinia chrysanthemi) enzyme

Abstract

The molecular model of Lycopersicon esculentum (tomato) pectin methylesterase (PME) was built by using the X-ray crystal structure of PME from the phytopathogenic bacterium Erwinia chrysanthemi as a template. The overall structure and the position of catalytically important residues (Asp132, Asp 153, and Arg 221, located at the bottom of the active site cleft) are conserved. Instead, loop regions forming the walls of the catalytic site are much shorter and form a less deep cleft, as already revealed by the carrot PME crystal structure. The protein inhibitor of pectin methylesterase (PMEI) isolated from kiwi fruit binds tomato PME with high affinity. Conversely, no complex formation between the inhibitor and PME from E. chrysanthemi is observed, and the activity of this enzyme is unaffected by the presence of the inhibitor. Fluorescence quenching experiments on tomato PME and on PME-PMEI complex suggest that tryptophanyl residues present in the active site region are involved in the interaction and that the inhibitor interacts with plant PME at the level of the active site. We also suggest that the more open active site cleft of tomato PME allows the interaction with the inhibitor. Conversely, the narrow and deep cleft of the active site of E. chrysanthemi PME hinders this interaction. The pH-dependent changes in fluorescence emission intensity observed in tomato PME could arise as the result of protonation of an Asp residue with unusually high pKa, thus supporting the hypothesis that Asp132 acts as acid/base in the catalytic cycle.

Inhibition of pectin methyl esterase activity by green tea catechins

“…Proteinaceous PME inhibitor (PMEI) from kiwi fruit inhibits by direct contact with the relatively wide active binding site cleft of PME , thus covering the binding site access point (D'Avino et al, 2003;Di Matteo et al, 2005). The FRET behavior exhibited in our samples ( Figure S3), in which emissions of fluorescence from the binding site tryptophan of PME are absorbed by EGCG and re-emitted at around 390 nm, suggests that the aromatic system in EGCG is interacting closely with tryptophan groups located on the PME molecule.…”

“…Small molecule inhibitors would be more tractable as applied enzyme inhibitors. Several proteinaceous inhibitors of pectin methyl esterase (termed PMEIs) have been identified (D'Avino et al, 2003;Giovane et al, 2004;Juge, 2006;Raiola et al, 2004;Wolf et al, 2003). However, to our knowledge, no small molecules have been implicated in inhibition of PME to date.…”

Structural Basis for the Interaction between Pectin Methylesterase and a Specific Inhibitor Protein

“…Trp223 of PME forms three contacts with its interacting counterpart, whereas Tyr135 forms only one contact; moreover, each of them forms a watermediated hydrogen bond. Upon formation of the complex with the inhibitor, Trp223 buries almost half of its solvent-exposed surface, and this explains the decrease of fluorescence observed in PME upon addition of the inhibitor (D'Avino et al, 2003). We can infer the mode of action of the inhibitor: on one hand, the inhibitor covers the active site cleft preventing the access of the substrate, and on the other hand, it prevents the interactions of Phe80, Tyr135, and Trp223 with the substrate.…”

“…The stability of the complex is pH dependent, being higher in acidic conditions, typical of the apoplastic environment, and decreasing drastically by raising the pH from 6.5 to 7.5; no formation of the complex occurs at pH 8.5 (D'Avino et al, 2003). The contact between the NE2 of His137 in PME and OG1 of Thr113 in the inhibitor (2.92 Å ) may be crucial for determining the strength of the interaction in this pH range.…”

Effect of Phenolic Acid on Antioxidant Activity of Wine and Inhibition of Pectin Methyl Esterase

“…It increased from 17.8 mM to 21.0 mM in the presence of 0.1% and 0.5% coumaric acid respectively (Table IV). Ki values of EGCG and PME inhibitor (PMEI) from kiwi to tomato PME were 420 μM (measured by cyano-acetate substrate) and 0.053 μM (using citrus pectin as the substrate) respectively 9,23 and to kiwi PME with an Ki of 0.22 μM (using citrus pectin as the substrate) 3 . The Ki values of gallic acid and coumaric acid were higher than that of EGCG and kiwi PMEI.…”