Experimental evidence for a hydride transfer mechanism in plant glycolate oxidase catalysis

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C3 and C4 plant species, we analyzed kinetic parameters of purified recombinant C3 (Arabidopsis thaliana) and C4 (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The 12C/13C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the 13C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C3 and C4 enzymes

Implication des complexes kinases SnRK1 ("SNF1-Related Kinase 1", AKIN) dans la signalisation par les sucres et la régulation du métabolisme chez Arabidopsis thaliana

Les kinases SNF1/AMPK/SnRK1 appartiennent à une famille de sérine/thréonine kinases remarquablement conservées au cours de l évolution. Elles agissent comme des senseurs de métabolites afin de constamment adapter le métabolisme de l organisme en fonction des ressources énergétiques. Chez la levure S. cerevisiae, le complexe kinase SNF1 est un élément central de la réponse à une carence en glucose. L AMPK, homologue de SNF1 chez les mammifères, joue un rôle central dans la régulation de l homéostasie énergétique. En comparaison à la levure et aux mammifères, peu de données sont disponibles sur les complexes kinase SnRK1 de plantes. L étude de ces kinases reste difficile de part leur implication dans de nombreuses fonctions et mécanismes moléculaires. Afin de préciser les rôles des deux kinases AKINa1 et AKINa2 de type SnRK1 chez A. thaliana, des plantes transgéniques surexprimant l ARNm de ces deux kinases ont été produites. L analyse des lignées 35S:AKINa1 par des approches génétiques, biochimiques, physiologiques et moléculaires ont permis d impliquer AKINa1 dans différentes voies de signalisation par les sucres et les hormones (ABA). De fortes modifications des teneurs en amidon, en sucres solubles ont été observées dans les lignées 35S:AKINa1. Ces plantes présentent également des modifications de l activité d enzymes essentielles comme la nitrate réductase ou l AGPase ainsi que la dérégulation de l expression de plusieurs gènes confirmant le rôle central des kinases SnRK1 dans la régulation du métabolisme. Des analyses préliminaires des lignées 35S:AKINa2 ont suggéré un rôle spécifique de de la kinase AKINa2 en réponse à un stress osmotique.The SNF1/AMPK/SnRK1 kinase is a subfamily of serine/threonine kinases highly conserved throughout evolution. They act as metabolite sensors to constantly adapt the metabolism to the level of energy. In the yeast Saccharomyces cerevisiae, the SNF1 kinase complex is a central component of the regulatory response to glucose starvation. The AMPK, the mammalian homologue of SNF1, plays a central role in the regulation of energy homeostatis at the cellular as well as the whole body levels. Compared to yeast or mammals, little is known about the SnRK1 complex in plant. Studying these kinases remains difficult because of their implication in multiple functions and molecular mechanisms. In order to precise the function of the two SnRK1 kinases AKINa1 and AKINa2 in Arabidopsis thaliana, transgenic plants overexpressing AKINa1 and AKINa2 have been produced. Genetic, biochemical, physiological and molecular analyses of these plants reveal that AKINa1 is implicated in different sugar and ABA signaling pathways. Strong modifications in starch and solubles sugars as well as in anthocyanins content were observed in the 35S:AKINa1 transgenic lines. They also present modifications of the activity of essential enzymes such as nitrate reductase or ADP-glucose pyrophosphorylase and of the expression of several genes regulated by sugars, confirming the central role of SnRK1 kinases in the regulation of metabolism. A preliminary analysis of 35S:AKINa2 transgenic lines suggests a specific implication of AKINa2 in the response to osmotic stress.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

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

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.</jats:p

Phosphomimetic T335D Mutation of Hydroxypyruvate Reductase 1 Modifies Cofactor Specificity and Impacts Arabidopsis Growth in Air

International audiencePhotorespiration is an essential process in oxygenic photosynthetic organisms triggered by the oxygenase activity of Rubisco. In peroxisomes, photorespiratory HYDROXYPYRUVATE REDUCTASE1 (HPR1) catalyzes the conversion of hydroxypyruvate to glycerate together with the oxidation of a pyridine nucleotide cofactor. HPR1 regulation remains poorly understood; however, HPR1 phosphorylation at T335 has been reported. By comparing the kinetic properties of phosphomimetic (T335D), nonphosphorylatable (T335A), and wild-type recombinant Arabidopsis (Arabidopsis thaliana) HPR1, it was found that HPR1-T335D exhibits reduced NADH-dependent hydroxypyruvate reductase activity while showing improved NADPH-dependent activity. Complementation of the Arabidopsis hpr1-1 mutant by either wild-type HPR1 or HPR1-T335A fully complemented the photorespiratory growth phenotype of hpr1-1 in ambient air, whereas HPR1-T335D-containing hpr1-1 plants remained smaller and had lower photosynthetic CO2 assimilation rates. Metabolite analyses indicated that these phenotypes were associated with subtle perturbations in the photorespiratory cycle of HPR1-T335D-complemented hpr1-1 rosettes compared to all other HPR1-containing lines. Therefore, T335 phosphorylation may play a role in the regulation of HPR1 activity in planta, although it was not required for growth under ambient air controlled conditions. Furthermore, improved NADP-dependent HPR1 activities in peroxisomes could not compensate for the reduced NADH-dependent HPR1 activit

The Impact of Photorespiratory Glycolate Oxidase Activity on Arabidopsis thaliana Leaf Soluble Amino Acid Pool Sizes during Acclimation to Low Atmospheric CO2 Concentrations

Photorespiration is a metabolic process that removes toxic 2-phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. It is essential for plant growth under ambient air, and it can play an important role under stress conditions that reduce CO2 entry into the leaf thus enhancing photorespiration. The aim of the study was to determine the impact of photorespiration on Arabidopsis thaliana leaf amino acid metabolism under low atmospheric CO2 concentrations. To achieve this, wild-type plants and photorespiratory glycolate oxidase (gox) mutants were given either short-term (4 h) or long-term (1 to 8 d) low atmospheric CO2 concentration treatments and leaf amino acid levels were measured and analyzed. Low CO2 treatments rapidly decreased net CO2 assimilation rate and triggered a broad reconfiguration of soluble amino acids. The most significant changes involved photorespiratory Gly and Ser, aromatic and branched-chain amino acids as well as Ala, Asp, Asn, Arg, GABA and homoSer. While the Gly/Ser ratio increased in all Arabidopsis lines between air and low CO2 conditions, low CO2 conditions led to a higher increase in both Gly and Ser contents in gox1 and gox2.2 mutants when compared to wild-type and gox2.1 plants. Results are discussed with respect to potential limiting enzymatic steps with a special emphasis on photorespiratory aminotransferase activities and the complexity of photorespiration.</jats:p

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

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

The Impact of Photorespiratory Glycolate Oxidase Activity on Arabidopsis thaliana Leaf Soluble Amino Acid Pool Sizes during Acclimation to Low Atmospheric CO2 Concentrations

International audiencePhotorespiration is a metabolic process that removes toxic 2-phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. It is essential for plant growth under ambient air, and it can play an important role under stress conditions that reduce CO2 entry into the leaf thus enhancing photorespiration. The aim of the study was to determine the impact of photorespiration on Arabidopsis thaliana leaf amino acid metabolism under low atmospheric CO2 concentrations. To achieve this, wild-type plants and photorespiratory glycolate oxidase (gox) mutants were given either short-term (4 h) or long-term (1 to 8 d) low atmospheric CO2 concentration treatments and leaf amino acid levels were measured and analyzed. Low CO2 treatments rapidly decreased net CO2 assimilation rate and triggered a broad reconfiguration of soluble amino acids. The most significant changes involved photorespiratory Gly and Ser, aromatic and branched-chain amino acids as well as Ala, Asp, Asn, Arg, GABA and homoSer. While the Gly/Ser ratio increased in all Arabidopsis lines between air and low CO2 conditions, low CO2 conditions led to a higher increase in both Gly and Ser contents in gox1 and gox2.2 mutants when compared to wild-type and gox2.1 plants. Results are discussed with respect to potential limiting enzymatic steps with a special emphasis on photorespiratory aminotransferase activities and the complexity of photorespiration

The long-term adaptive response of air-grown Arabidopsis ggt1 photorespiratory mutants involves a drastic reduction in leaf RuBisCO content

International audienc

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1

International audienceTo measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni 2+ -NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase and hydroxypyruvate reductase) from E. coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD