Radical cation scavenging activity of berberine bridge enzyme-like oligosaccharide oxidases acting on short cell wall fragments.

Oligogalacturonide-oxidases (OGOXs) and cellodextrin-oxidase (CELLOX) are plant berberine bridge enzyme-like oligosaccharide-oxidases (OSOXs) that oxidize, respectively, oligogalacturonides (OGs) and cellodextrins (CDs), thereby inactivating their elicitor nature and concomitantly releasing H2O2. Little is known about the physiological role of OSOX activity. By using an ABTS·+-reduction assay, we identified a novel reaction mechanism through which the activity of OSOXs on cell wall oligosaccharides scavenged the radical cation ABTS·+ with an efficiency dependent on the type and length of the oxidized oligosaccharide. In contrast to the oxidation of longer oligomers such as OGs (degree of polymerization from 10 to 15), the activity of OSOXs on short galacturonan- and cellulose-oligomers (degree of polymerization ≤ 4) successfully counteracted the radical cation-generating activity of a fungal laccase, suggesting that OSOXs can generate radical cation scavenging activity in the apoplast with a power proportional to the extent of degradation of the plant cell wall, with possible implications for redox homeostasis and defense against oxidative stress.

Molecular dynamics simulations and kinetic measurements provide insights into the structural requirements of substrate size-dependent specificity of oligogalacturonide oxidase 1 (OGOX1).

Oligogalacturonides (OGs) are pectin fragments released from the breakdown of the homogalacturonan during pathogenesis that act as Damage-Associated Molecular Patterns. OG-oxidase 1 (OGOX1) is an Arabidopsis berberine bridge enzyme-like (BBE-l) oligosaccharide oxidase that oxidizes OGs, impairing their elicitor activity and concomitantly releasing H2O2. The OG-oxidizing activity of OGOX1 is markedly pH-dependent, with optimum pH around 10, and is higher towards OGs with a degree of polymerization higher than two. Here, the molecular determinants of OGOX1 responsible for the binding of OGs with different lengths have been investigated through molecular dynamics simulations and enzyme kinetics studies. OGOX1 was simulated in complex with OGs with different degree of polymerization such as di-, tri-, tetra- and penta-galacturonide, in water solution at alkaline pH. Our simulations revealed that, among the four OGOX1/OG combinations, the penta-galacturonide (OG5) showed the best conformation in the active site to be efficiently oxidized by OGOX1. The optimal conformation can be stabilized by salt-bridges formed between the carboxyl groups of OG5 and five positively charged amino acids of OGOX1, highly conserved in all OGOX paralogs. Our results suggest that these interactions limit the mobility of OG5 as well as longer OGs, contributing to maintain the terminal monomer of OGs in the optimal orientation in order to be oxidized by the enzyme. In accordance with these results, the enzyme efficiency (Kcat/KM) of OGOX1 on OG5 (40.04) was found to be significantly higher than that on OG4 (13.05) and OG3 (0.6).

Characterization of two 1,3-ß-glucan-modifying enzymes from Penicillium sumatraense reveals new insights into 1,3-ß-glucan metabolism of fungal saprotrophs.

BACKGROUND: 1,3-ß-glucan is a polysaccharide widely distributed in the cell wall of several phylogenetically distant organisms, such as bacteria, fungi, plants and microalgae. The presence of highly active 1,3-ß-glucanases in fungi evokes the biological question on how these organisms can efficiently metabolize exogenous sources of 1,3-ß-glucan without incurring in autolysis. RESULTS: To elucidate the molecular mechanisms at the basis of 1,3-ß-glucan metabolism in fungal saprotrophs, the putative exo-1,3-ß-glucanase G9376 and a truncated form of the putative glucan endo-1,3-ß-glucosidase (ΔG7048) from Penicillium sumatraense AQ67100 were heterologously expressed in Pichia pastoris and characterized both in terms of activity and structure. G9376 efficiently converted laminarin and 1,3-ß-glucan oligomers into glucose by acting as an exo-glycosidase, whereas G7048 displayed a 1,3-ß-transglucanase/branching activity toward 1,3-ß-glucan oligomers with a degree of polymerization higher than 5, making these oligomers more recalcitrant to the hydrolysis acted by exo-1,3-ß-glucanase G9376. The X-ray crystallographic structure of the catalytic domain of G7048, solved at 1.9 Å of resolution, consists of a (ß/α)8 TIM-barrel fold characteristic of all the GH17 family members. The catalytic site is in a V-shaped cleft containing the two conserved catalytic glutamic residues. Molecular features compatible with the activity of G7048 as 1,3-ß-transglucanase are discussed. CONCLUSIONS: The antagonizing activity between ΔG7048 and G9376 indicates how opportunistic fungi belonging to Penicillium genus can feed on substrates similar for composition and structure to their own cell wall without incurring in a self-deleterious autohydrolysis.

Berberine Bridge Enzyme-like Oligosaccharide Oxidases Act as Enzymatic Transducers Between Microbial Glycoside Hydrolases and Plant Peroxidases.

Oligogalacturonide (OG)-oxidase 1 (OGOX1) and cellodextrin (CD)-oxidase (CELLOX) are plant berberine bridge enzyme-like oligosaccharide oxidases that oxidize OGs and CDs, cell-wall fragments with the nature of damage-associated molecular patterns. The oxidation of OGs and CDs attenuates their elicitor activity and concomitantly releases H2O2. By using a multiple enzyme-based assay, we demonstrate that the H2O2 generated downstream of the combined action between a fungal polygalacturonase and OGOX1 or an endoglucanase and CELLOX can be directed by plant peroxidases (PODs) either towards a reaction possibly involved in plant defense, such as the oxidation of monolignol or a reaction possibly involved in a developmental event, such as the oxidation of auxin (indole-3-acetic acid), pointing to OGOX1 and CELLOX as enzymatic transducers between microbial glycoside hydrolases and plant PODs. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A molecular dynamics-guided mutagenesis identifies two aspartic acid residues involved in the pH-dependent activity of OG-OXIDASE 1.

During the infection, plant cells secrete different OG-oxidase (OGOX) paralogs, defense flavoproteins that oxidize the oligogalacturonides (OGs), homogalacturonan fragments released from the plant cell wall that act as Damage Associated Molecular Patterns. OGOX-mediated oxidation inactivates their elicitor nature, but on the other hand makes OGs less hydrolysable by microbial endo-polygalacturonases (PGs). Among the different plant defense responses, apoplastic alkalinization can further reduce the degrading potential of PGs by boosting the oxidizing activity of OGOXs. Accordingly, the different OGOXs so far characterized showed an optimal activity at pH values greater than 8. Here, an approach of molecular dynamics (MD)-guided mutagenesis succeeded in identifying the amino acids responsible for the pH dependent activity of OGOX1 from Arabidopsis thaliana. MD simulations indicated that in alkaline conditions (pH 8.5), the residues Asp325 and Asp344 are engaged in the formation of two salt bridges with Arg327 and Lys415, respectively, at the rim of enzyme active site. According to MD analysis, the presence of such ionic bonds modulates the size and flexibility of the cavity used to accommodate the OGs, in turn affecting the activity of OGOX1. Based on functional properties of the site-directed mutants OGOX1.D325A and OGOX.D344A, we demonstrated that Asp325 and Asp344 are major determinants of the alkaline-dependent activity of OGOX1.