Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of <i>Arabidopsis thaliana</i>

Rippert, Pascal; Matringe, Michel

doi:10.1046/j.1432-1033.2002.03172.x

Cited by 75 publications

(109 citation statements)

References 36 publications

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“…TYRA protein of an apparent molecular mass of 65 to 67 kD. These results are in agreement with our previous biochemical studies carried out on purified recombinant TYRAAt1 and TYRAAt2 overproduced in E. coli cells (Rippert and Matringe, 2002b). In this previous study, the purified recombinant TYRAAt2 eluted in the void volume of the S200 gel filtration column, whereas the purified recombinant TYRAAt1 eluted with a mobility consistent with a protein of 66 kD.…”

Section: Analysis Of Tyra Activity From Arabidopsis Leavessupporting

confidence: 92%

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Tyrosine and Phenylalanine Are Synthesized within the Plastids in Arabidopsis

et al. 2009

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While the presence of a complete shikimate pathway within plant plastids is definitively established, the existence of a cytosolic postchorismate portion of the pathway is still debated. This question is alimented by the presence of a chorismate mutase (CM) within the cytosol. Until now, the only known destiny of prephenate, the product of CM, is incorporation into tyrosine (Tyr) and/or phenylalanine (Phe). Therefore, the presence of a cytosolic CM suggests that enzymes involved downstream of CM in Tyr or Phe biosynthesis could be present within the cytosol of plant cells. It was thus of particular interest to clarify the subcellular localization of arogenate dehydrogenases (TYRAs) and arogenate dehydratases (ADTs), which catalyze the ultimate steps in Tyr and Phe biosynthesis, respectively. The aim of this study was to address this question in Arabidopsis (Arabidopsis thaliana) by analysis of the subcellular localization of the two TYRAAts and the six AtADTs. This article excludes the occurrence of a spliced TYRAAt1 transcript encoding a cytosolic TYRA protein. Transient expression analyses of TYRA-and ADT-green fluorescent protein fusions reveal that the two Arabidopsis TYRA proteins and the six ADT proteins are all targeted within the plastid. Accordingly, TYRA and ADT proteins were both immunodetected in the chloroplast soluble protein fraction (stroma) of Arabidopsis. No TYRA or ADT proteins were immunodetected in the cytosol of Arabidopsis cells. Taken together, all our data exclude the possibility of Tyr and/or Phe synthesis within the cytosol, at least in green leaves and Arabidopsis cultured cells.

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Section: Analysis Of Tyra Activity From Arabidopsis Leavessupporting

confidence: 92%

“…Arogenate was synthesized enzymatically from prephenate and Asp as previously described (Rippert and Matringe, 2002b …”

Section: Methodsmentioning

confidence: 99%

Tyrosine and Phenylalanine Are Synthesized within the Plastids in Arabidopsis

et al. 2009

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“…While there was no Tyr depletion, this is not necessarily expected. First, Phe and Tyr synthesis both branch from L-arogenate and are subject to feedback inhibition and activation loops that tend to dampen the impact of perturbations (Figure 8) (Jung et al, 1986;Siehl and Conn, 1988;Rippert and Matringe, 2002;Tzin and Galili, 2010). Second, only the chloroplastic Tyr pool would be immediately affected by loss of AAH, and this pool is presumably only part of the total free Tyr pool that was measured.…”

Section: Moss Aah Mediates Significant Phe To Tyr Flux In Vivomentioning

confidence: 99%

“…The AAH short circuit potentially confers three unique features on aromatic metabolism. These features are of special interest in gymnosperms, in which ;30% of the carbon fixed in photosynthesis courses down the Phe branch en route to lignin, the flux down the Tyr branch being far smaller (Rippert and Matringe, 2002). First, the short circuit provides an alternative biosynthetic path to Tyr.…”

Section: Plant Aah: a Short Circuit Between Two Major Branches Of Aromentioning

confidence: 99%

Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts

et al. 2010

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Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of fulllength AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism.

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“…As a check on the quality of the genomic DNA, an internal fragment of the Arabidopsis arogenate dehydrogenase gene (GenBank accession no. AF434682) 25) was amplified with primers 5 0 -TGGAT-CGCAATTCGTGACGCATAC-3 0 and 5 0 -AAAGTCG-ACTTAAGATGATGATGATGATGATC-3 0 . For RNA analysis, total RNA was extracted from Arabidopsis transformants with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and 5 mg of the RNA was subjected to RNA gel-blot hybridization.…”

Section: Methodsmentioning

confidence: 99%