Enzymatic Properties of Recombinant Phospho-Mimetic Photorespiratory Glycolate Oxidases from <i>Arabidopsis thaliana</i> and <i>Zea mays</i>

• Main Authors: Mathieu Jossier, Yanpei Liu, Sophie Massot, Michael Hodges
• Format: Article
• Language: English
• Published: MDPI AG 2019-12-01
• Series: Plants
• Subjects: <i>arabidopsis thaliana</i>; glycolate oxidase; photorespiration; protein phosphorylation; <i>zea mays</i>
• Online Access: https://www.mdpi.com/2223-7747/9/1/27

In photosynthetic organisms, the photorespiratory cycle is an essential pathway leading to the recycling of 2-phosphoglycolate, produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase, to 3-phosphoglycerate. Although photorespiration is a widely studied process, its regulation remains poorly understood. In this context, phosphoproteomics studies have detected six phosphorylation sites associated with photorespiratory glycolate oxidases from <i>Arabidopsis thaliana</i> (<i>At</i>GOX1 and <i>At</i>GOX2). Phosphorylation sites at T4, T158, S212 and T265 were selected and studied using Arabidopsis and maize recombinant glycolate oxidase (GOX) proteins mutated to produce either phospho-dead or phospho-mimetic enzymes in order to compare their kinetic parameters. Phospho-mimetic mutations (T4D, T158D and T265D) led to a severe inhibition of GOX activity without altering the K_M glycolate. In two cases (T4D and T158D), this was associated with the loss of the cofactor, flavin mononucleotide. Phospho-dead versions exhibited different modifications according to the phospho-site and/or the GOX mutated. Indeed, all T4V and T265A enzymes had kinetic parameters similar to wild-type GOX and all T158V proteins showed low activities while S212A and S212D mutations had no effect on <i>At</i>GOX1 activity and <i>At</i>GOX2/<i>Zm</i>GO1 activities were 50% reduced. Taken together, our results suggest that GOX phosphorylation has the potential to modulate GOX activity.