A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids

Authors: ZHANG, BIXIA - Washington State University; VERMERRIS, WILFRED - University Of Florida; Sattler, Scott; KANG, CHULHEE - Washington State University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2022
Publication Date: 5/1/2023
Citation: Zhang, B., Vermerris, W., Sattler, S.E., Kang, C. 2023. A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids. Nature Communications. 192(1):102-118. https://doi.org/10.1093/plphys/kiac604.
DOI: https://doi.org/10.1093/plphys/kiac604

Interpretive Summary: Hydrogen peroxide (H2O2) is produced in cells, either as by-products of enzymatic reactions or as part of a defense response to pathogens and pests. Ascorbate peroxidase (APX) is considered a key enzyme for controlling the levels of this toxic compound in higher plants using ascorbate (Vitamin C). The structure of the sorghum ascorbate peroxidase was determined through X-ray crystallography. While the overall structure of SbAPX was similar to the solved structures of other APX family members, SbAPX uniquely bound four molecules of ascorbate rather than a single ascorbate molecule as previously shown for this enzyme from other plants. The four binding sites differed in their ability to bind ascorbate. Observed for the first time, APX catalyzed the H2O2-dependent oxidation of ascorbate to the oxidized form dehydroascorbic acid, which indicates two successive electrons were transferred from the APX-bound ascorbate. In addition, several other substrates were also tested, which showed the enzyme was able to act upon other organic compounds like previously shown. This enzyme may participate in other metabolic pathways in addition to controlling the cellular levels of hydrogen peroxide in sorghum and other plants.

Technical Abstract: In planta, the reactive oxygen species (ROS) hydrogen peroxide (H2O2) is produced in various cellular compartments, either as by-products of enzymatic reactions or as part of a defense response to pathogens and pests. APX is considered a key antioxidant enzyme in higher plants, scavenging H2O2 with ascorbate to form H2O and two monodehydroascorbate radicals. Here we report the crystal structures of cytosolic ascorbate peroxidase from Sorghum bicolor (SbAPX; Sobic.001G410200). While the overall structure of SbAPX was similar to the solved structures of other APX family members, SbAPX uniquely displayed four rather than one bound ascorbate molecule. In addition to the '-heme edge binding pocket identified in other APX enzymes, ascorbates were bound at the '-meso site and two solvent-exposed binding pockets. The four pockets differed in their degree of hydrophobicity, and, consequently, in the affinity for the substrate. Consistent with the presence of multiple binding sites, our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Variation in the amino acid composition of each substrate-binding pocket, especially at the '-meso site, could affect specificity of binding for substrates other than ascorbate. Bound ascorbate at two surface sites established an intricate proton network with ascorbate at the '-heme edge and '-meso, offering effective reduction of cytotoxic H2O2. For the first time, we have observed that the H2O2-dependent oxidation of ascorbate by APX produces a C2-hydrated bicyclic hemiketal form of dehydroascorbic acid bound at the '-heme edge, which indicates two successive electron transfers from a bound ascorbate. In addition, several other substrates, including p-coumaric acid, were tested to assess the substrate specificity of SbAPX. Only d-meso site was shared with those organic compounds for the potential radicalization reaction, as previously noted. Based on detailed structural and kinetic analyses, as well as that of closely related APX enzymes, the critical residues in each substrate-binding site of SbAPX and detailed catalytic mechanism of SbAPX are proposed.