Two genes in <i>Arabidopsis thaliana</i> encoding GDP‐<scp>l</scp>‐galactose phosphorylase are required for ascorbate biosynthesis and seedling viability

Dowdle, John; Ishikawa, Takahiro; Gatzek, Stephan; Rolinski, Susanne; Smirnoff, Nicholas

doi:10.1111/j.1365-313x.2007.03266.x

Cited by 398 publications

(536 citation statements)

References 79 publications

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“…The presence of alternative biosynthesis or recycling pathways for AsA that are independent of mannose would provide an explanation for this paradox (48). However, a recent analysis of the vtc2 vtc5 double mutants has revealed that the GDPmannose pathway is the only significant source of AsA in Arabidopsis seedlings (49). Another potential explanation would be that pmm-12 prioritizes the flux through GDP-mannose toward AsA biosynthesis at the expense of glycosylation.…”

Section: Map-based Cloning Of the Pmm-12mentioning

confidence: 91%

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Hoeberichts

Vaeck

Kiddle

et al. 2008

Journal of Biological Chemistry

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Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16 -18°C but died within several days after transfer to 28°C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat /K m ). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

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Section: Map-based Cloning Of the Pmm-12mentioning

confidence: 91%

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Hoeberichts

Vaeck

Kiddle

et al. 2008

Journal of Biological Chemistry

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“…Here we find that vtc2-2 and vtc2-3 mutant proteins are nearly devoid of enzymatic activity and that a VTC2 homologous enzyme, termed VTC5 (16), has nearly identical enzymatic characteristics. We find that VTC2 activity and formation of a covalent guanylylated intermediate both depend on His-238.…”

mentioning

confidence: 89%

“…We found that At5g55120 encodes a GDP-L-Gal phosphorylase whose biochemical properties closely resemble those of VTC2 (Table 2) and which might, thus, account for the residual vitamin C levels found in vtc2 mutants. During the course of these studies Dowdle et al (16) also overexpressed At5g55120 and characterized the gene product, which they named VTC5, as a GDP-L-Gal phosphorylase.…”

Section: Vtc2 Point Mutants Lackmentioning

confidence: 99%

A Second GDP-l-galactose Phosphorylase in Arabidopsis en Route to Vitamin C

Linster¹,

Adler²,

Webb³

et al. 2008

Journal of Biological Chemistry

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The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the SmirnoffWheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.Vitamin C (L-ascorbic acid) is the most abundant soluble antioxidant in plants, in which it plays crucial roles in protection against oxidative damage and is used as a cofactor for several enzymes. It is synthesized via reactions initially proposed by Wheeler et al. (1) that are distinct from those used in vitamin C biosynthesis in animals. Although the Smirnoff-Wheeler pathway, arising from GDP-D-mannose and involving L-galactose formation, might not be the only route to vitamin C biosynthesis in plants (alternative pathways arising from myo-inositol and methylgalacturonate or involving L-gulose formation have been proposed; for reviews, see Refs. 2 and 3), it is undoubtedly the pathway that has received the strongest biochemical and genetic support in recent years (4 -9).Of the four loci (VTC1-4) that have been found to be mutated in vitamin C-deficient Arabidopsis thaliana plants (10, 11), three are now known to encode enzymes involved in the Smirnoff-Wheeler pathway. VTC1 encodes GDP-mannose pyrophosphorylase (12), whereasVTC4 encodes L-Gal-1-P 3 phosphatase (8). In 2007 the VTC2 gene product was identified as the enzyme that produces L-Gal-1-P from GDP-L-galactose, which completed the characterization of the 10 enzymatic steps leading from D-glucose to L-ascorbic acid (13,14). VTC2 is a member of the D-galactose-1-phosphate ...

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“…In this context, the widespread presence of multiple copies of putative AO-encoding genes in virtually all plant taxa, as evidenced by EST analysis, seems inexplicable. No surprise that AO has been defined a 'mysterious enzyme' [6].…”

Section: Introductionmentioning

confidence: 99%

Ascorbate oxidase: The unexpected involvement of a ‘wasteful enzyme’ in the symbioses with nitrogen-fixing bacteria and arbuscular mycorrhizal fungi

Balestrini¹,

Ott²,

Güther³

et al. 2012

Plant Physiology and Biochemistry

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Two genes in Arabidopsis thaliana encoding GDP‐l‐galactose phosphorylase are required for ascorbate biosynthesis and seedling viability

Cited by 398 publications

References 79 publications

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

A Second GDP-l-galactose Phosphorylase in Arabidopsis en Route to Vitamin C

Ascorbate oxidase: The unexpected involvement of a ‘wasteful enzyme’ in the symbioses with nitrogen-fixing bacteria and arbuscular mycorrhizal fungi

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