Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins

Abstract

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.

Phenotypic Investigation and RNA-seq of KN1 Involved in Leaf Angle Formation in Maize (Zea mays L.)

“…Studies on Arabidopsis mutants with low flavonoid content have shown that auxin transport levels are inhibited by flavonoids [ 55 ]. XTH and PGI participate in the cell wall remodeling process by catalyzing glycosyl transfer [ 56 ] and inhibiting the activity of polygalacturonase (PG) [ 57 ], respectively, and then participate in the control of plant morphology by affecting cell elongation. According to the expression of the above related genes in B73 and kn1 after treatment with different exogenous phytohormones, we summarized the possible molecular model of exogenous phytohormones involved in the regulation of LA ( Figure 10 ).…”

TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells

“…MSC surface wall powders were resuspended in 1 mL distilled water for 1 h. An aliquot (450 μL) was mixed with 50 μL of 500 mM sodium acetate buffer pH 5.0 and polysaccharides were hydrolyzed for 24 h at 40°C under 500 rpm shaking with either a pectin lyase (PL) specific for methylesterified HGs, 43 a polygalacturonase (PG) 71 specific for non-methylesterified HGs or a rhamnogalacturonase (RG, Swiss-Prot Q00018, provided by Novozymes, Copenhagen, Denmark) specific for the RGI backbone. 50 Resulting OGA digests were filtered on 0.45 μm and analyzed by IP-RP-UHPC-MS. Acquisitions were performed on a Select Series Cyclic IMS (Waters, Wilmslow, UK) coupled with a UHPLC system (Acquity H-Class Plus, Waters, Manchester, UK).…”

TBL38 is an atypical homogalacturonan acetylesterase with a peculiar cell wall microdomain localization in Arabidopsis seed mucilage secretory cells

“…MSC surface wall powders were resuspended in 1 mL distilled water for 1 h. An aliquot (450 µL) was mixed with 50 µL of 500 mM sodium acetate buffer pH 5.0 and polysaccharides were hydrolyzed for 24 h at 40°C under 500 rpm shaking with either a pectin lyase (PL) specific for methylesterified HGs (Ralet et al, 2012), a polygalacturonase (PG) (Safran et al, 2023) specific for non-methylesterified HGs or a rhamnogalacturonase (RG, Swiss-Prot Q00018, provided by Novozymes, Copenhagen, Denmark) specific for the RGI backbone (Ralet et al, 2010). Resulting OGA digests were filtered on 0.45 µm and analyzed by IP-RP-UHPC-MS. Acquisitions were performed on a Select Series Cyclic IMS (Waters, Wilmslow, UK) coupled with a UHPLC system (Acquity H-Class Plus, Waters, Manchester, UK).…”