Enzymatic Properties of Recombinant Phospho-Mimetic Photorespiratory Glycolate Oxidases from Arabidopsis thaliana and Zea mays.

Mathieu Jossier,Yanpei Liu,Michael Hodges,Sophie Massot

doi:10.3390/plants9010027

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 (AtGOX1 and AtGOX2). 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 AtGOX1 activity and AtGOX2/ZmGO1 activities were 50% reduced. Taken together, our results suggest that GOX phosphorylation has the potential to modulate GOX activity.

Highlights

Photorespiration begins with the fixation of O2 to ribulose-1,5-bisphosphate by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) leading to the formation of one molecule of 2-phosphoglycolate (2PG) and one molecule of 3-phosphoglycerate (3PGA). 2PG is metabolized to produce 3PGA by the photorespiratory cycle which occurs in four subcellular compartments, and involves eight core enzymes and several transporters [1]
Six phosphopeptides were found with six different phosphorylated residues; T4, T155, T158, S212, T265 phosphopeptides associated with both AtGOX1 and AtGOX2 and T355 that was in a peptide associated only with AtGOX1 (Table 1)
glycolate oxidase (GOX) phosphorylation could be important since several phospho-sites have been reported and the phosphorylated residues have been conserved during the phosphorylated [24]

Summary

Introduction

Photorespiration begins with the fixation of O2 to ribulose-1,5-bisphosphate by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) leading to the formation of one molecule of 2-phosphoglycolate (2PG) and one molecule of 3-phosphoglycerate (3PGA). 2PG is metabolized to produce 3PGA by the photorespiratory cycle which occurs in four subcellular compartments (chloroplasts, peroxisomes, mitochondria and cytosol), and involves eight core enzymes and several transporters [1]. CO2 assimilation due to competition at the RuBisCO active site, and it releases assimilated carbon and nitrogen as CO2 and ammonium that have to be either reassimilated at an energetic cost or lost This has led to efforts to minimize the negative effects of photorespiration to improve plant yield by producing plants containing a chloroplastic bypass to metabolize photorespiratory glycolate (the most recent examples being [2,3]). Plants 2020, 9, 27 from toxic 2PG [4] is normally metabolized in peroxisomes by glycolate oxidase (GOX), a flavin mononucleotide (FMN) containing enzyme that catalyzes the transformation of glycolate to glyoxylate with the production of hydrogen peroxide [5].

Methods

Results

Conclusion