Activity of an atypical Arabidopsis thaliana pectin methylesterase

Abstract

An Arabidopsis thaliana pectin methylesterase that was not predicted to contain any signaling sequence was produced in E. coli and purified using a His tag added at its N-terminus. The enzyme demethylesterified Citrus pectin with a Km of 0.86 mg/ml. The enzyme did not require salt for activity and was found to be relatively temperature-sensitive. The precipitation of enzyme-treated pectin by CaCl2 suggested that the enzyme had a blockwise mode of pectin demethylesterification. A purified kiwi (Actinidia chinensis) pectin methylesterase inhibitor had no effect on the activity of the enzyme whereas it strongly inhibited a flax pectin methylesterase. A model of the protein structure revealed that an extra amino acid sequence in this particular Arabidopsis pectin methylesterase could form a ss-strand outside the core structure, which might be preventing the inhibitor from binding the protein.

Plant pectin acetylesterase structure and function: new insights from bioinformatic analysis

“…The absence of a predicted signaling sequence for three AtPAEs, AtPAE2, AtPAE4 and AtPAE5, indicates that some Arabidopsis PAEs without a signal peptide could have a functional activity. Similar results have been observed with several PMEs from distinct plant species and particularly for AtPME31, which has no signaling sequence but is active and cannot be inhibited by the kiwi pectin methylesterase inhibitor, a strong PME inhibitor [56]. 3D homology modeling of AtPME31 revealed an external loop.…”

Pectin methylesterase and its proteinaceous inhibitor: a review

“…62,69,212 Notably, kiwi PMEI has no effect on the activity of an atypical plant PME recently purified from A. thaliana. 211 A similar specificity (i.e., active against various plant PMEs but insensitive to microbial PMEs and invertase) was observed for the two recombinant Arabidopsis PMEIs. 110,200 …”

“…It is not active towards other (poly)saccharide-degrading enzymes, such as PG, amylase and invertase. 100,101 Kiwi PMEI inhibits successfully the activity of a wide range of plant PMEs (e.g., orange, apple, tomato, kiwi, kaki, potato, apricot, banana, cucumber, strawberry and flax), 17,100,102,204,211 whereas it is ineffective against all microbial PMEs assayed so far, fungal as well as bacterial. 62,69,212 Notably, kiwi PMEI has no effect on the activity of an atypical plant PME recently purified from A. thaliana.…”

“…63 Moreover, the lack of inhibition of an atypical plant PME from Arabidopsis by kiwi PMEI is possibly due to a b-strand outside the core structure of PME, matching the position of the external loop found in E. chrysanthemi PME. 211 …”

Pectinmethylesterases (PME) and Pectinmethylesterase Inhibitors (PMEI) Enriched during Phloem Fiber Development in Flax (Linum usitatissimum)

“…On the other hand, the heterologous expression of PMEs has produced less consistent results. The complete proteins of the type-2 PMEs QUARTET1 [6] and AtPME31 [7] were successfully expressed in E. coli strains BL21(DE3) and JM101, respectively. However, the mature portion (removing signal peptide and pro-region) of a type-1 PME (At1g11580) was expressed in E. coli strain M15 but was not functional compared to the native protein from Arabidopsis [8].…”