**Mathieu Jossier, Yanpei Liu, Sophie Massot and Michael Hodges \***

Institute of Plant Sciences Paris-Saclay, CNRS, Université Paris-Sud, INRA, Université d'Evry, Université Paris-Diderot, Université Paris-Saclay, 91405 Orsay CEDEX, France;

mathieu.jossier@ips2.universite-paris-saclay.fr (M.J.); yanpei.liu@ips2.universite-paris-saclay.fr (Y.L.);

sophie.massot@ips2.universite-paris-saclay.fr (S.M.)

**\*** Correspondence: michael.hodges@cnrs.fr

Received: 22 November 2019; Accepted: 21 December 2019; Published: 24 December 2019

**Abstract:** 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 *Arabidopsis thaliana* (*At*GOX1 and *At*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 KM 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 *At*GOX1 activity and *At*GOX2/*Zm*GO1 activities were 50% reduced. Taken together, our results suggest that GOX phosphorylation has the potential to modulate GOX activity.

**Keywords:** *Arabidopsis thaliana*; glycolate oxidase; photorespiration; protein phosphorylation; *Zea mays*
