Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of two Arabidopsis thaliana polygalacturonases, POLYGALACTURONASE LATERAL ROOT (PGLR) and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are co-expressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-Inhibiting Proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate-binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.
Efficient design of functional proteins with higher thermal stability remains challenging especially for highly diverse sequence variants. Considering the evolutionary pressure on protein folds, sequence design optimizing evolutionary fitness could help designing folds with higher stability. Using a generative evolution fitness model trained to capture variation patterns in natural sequences, we designed artificial sequences of a proteinaceous inhibitor of pectin methylesterase enzymes. These inhibitors have considerable industrial interest to avoid phase separation in fruit juice manufacturing or reduce methanol in distillates, averting chromatographic passages triggering unwanted aroma loss. Six out of seven designs with up to 30 % divergence to other inhibitor sequences are functional and two have improved thermal stability. This method can improve protein stability expanding functional protein sequence space, with traits valuable for industrial applications and scientific research.
The fine-tuning of pectins by polygalacturonases (PGs) plays a key role in modulating plant cell wall chemistry and mechanics, impacting plant development. The high number of plant PG isoforms and their absence of inhibition by endogenous PG-Inhibiting Proteins (PGIPs) question the regulation of pectin depolymerization during development. Our understanding of the diversity and of the regulation of plant PGs has been impaired by the lack of protein structures. Here we resolved the crystal structures of two PGs from Arabidopsis, PGLR and ADPG2, whose expression overlap in roots. By combining molecular dynamic simulations, analysis of enzymes kinetics and hydrolysis products we determined why plant PGs are not inhibited by PGIPs. We further showed that subtle differences in PGLR and ADPG2 structures translated into distinct dynamics and processivities, leading to peculiar effects on root development, as determined by exogenous applications of enzymes.
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