A tyrosine aminotransferase involved in tocopherol synthesis in Arabidopsis

The metabolic function of the predicted Arabidopsis tyrosine aminotransferase (TAT) encoded by the At5g53970 gene was studied using two independent knock-out mutants. Gas chromatography-mass spectrometry based metabolic profiling revealed a specific increase in tyrosine levels, supporting the proposed function of At5g53970 as a tyrosine-specific aminotransferase not involved in tyrosine biosynthesis, but rather in utilization of tyrosine for other metabolic pathways. The TAT activity of the At5g53970-encoded protein was verified by complementation of the Escherichia coli tyrosine auxotrophic mutant DL39, and in vitro activity of recombinantly expressed and purified At5g53970 was found to be specific for tyrosine. To investigate the physiological role of At5g53970, the consequences of reduction in tyrosine utilization on metabolic pathways having tyrosine as a substrate were analysed. We found that tocopherols were substantially reduced in the mutants and we conclude that At5g53970 encodes a TAT important for the synthesis of tocopherols in Arabidopsis.     

Biochemical and phylogenetic characterization of a novel diaminopimelate biosynthesis pathway in prokaryotes identifies a diverged form of LL-diaminopimelate aminotransferase

A variant of the diaminopimelate (DAP)-lysine biosynthesis pathway uses an LL-DAP aminotransferase (DapL, EC 2.6.1.83) to catalyze the direct conversion of L-2,3,4,5-tetrahydrodipicolinate to LL-DAP. Comparative genomic analysis and experimental verification of DapL candidates revealed the existence of two diverged forms of DapL (DapL1 and DapL2). DapL orthologs were identified in eubacteria and archaea. In some species the corresponding dapL gene was found to lie in genomic contiguity with other dap genes, suggestive of a polycistronic structure. The DapL candidate enzymes were found to cluster into two classes sharing approximately 30% amino acid identity. The function of selected enzymes from each class was studied. Both classes were able to functionally complement Escherichia coli dapD and dapE mutants and to catalyze LL-DAP transamination, providing functional evidence for a role in DAP/lysine biosynthesis. In all cases the occurrence of dapL in a species correlated with the absence of genes for dapD and dapE representing the acyl DAP pathway variants, and only in a few cases was dapL coincident with ddh encoding meso-DAP dehydrogenase. The results indicate that the DapL pathway is restricted to specific lineages of eubacteria including the Cyanobacteria, Desulfuromonadales, Firmicutes, Bacteroidetes, Chlamydiae, Spirochaeta, and Chloroflexi and two archaeal groups, the Methanobacteriaceae and Archaeoglobaceae.     

Dual diaminopimelate biosynthesis pathways in Bacteroides fragilis and Clostridium thermocellum

Bacteroides fragilis and Clostridium thermocellum were recently found to synthesize diaminopimelate (DAP) by way of LL-DAP aminotransferase. Both species also contain an ortholog of meso-diaminopimelate dehydrogenase (Ddh), suggesting that they may have redundant pathways for DAP biosynthesis. The B. fragilis Ddh ortholog shows low homology with other examples of Ddh and this species belongs to a phylum, the Bacteriodetes, not previously known to contain this enzyme. By contrast, the C. thermocellum ortholog is well conserved with known examples of Ddh. Using in vitro and in vivo assays both the B. fragilis and C. thermocellum enzymes were found to be authentic examples of Ddh, displaying kinetic properties typical of this enzyme. The result indicates that B. fragilis contains a sequence diverged form of Ddh. Phylogenomic analysis of the microbial genome database revealed that 77% of species with a Ddh ortholog also contain a second pathway for DAP biosynthesis suggesting that Ddh evolved as an ancillary mechanism for DAP biosynthesis.     

Characterization of homogentisate prenyltransferases involved in plastoquinone-9 and tocochromanol biosynthesis

A cDNA of Chlamydomonas reinhardtii encoding a plastidial homogentisate prenyltransferase was identified. Functional expression studies in Escherichia coli revealed that the enzyme possessed properties similar to the prenyltransferase of Arabidopsis thaliana encoded by At3g11950 but different from the phytyltransferases of A. thaliana and Synechocystis. Unlike the phytyltransferases, the C. reinhardtii and the respective A. thaliana enzyme showed highest activities with solanesyl diphosphate, but were hardly active with phytyl diphosphate. Hence, these data provide evidence that the latter represent homogentisate solanesyltransferases involved in plastoquinone-9 biosynthesis. Overexpression of At3g11950 in A. thaliana, however, suggests that the solanesyltransferase can affect tocopherol biosynthesis as well.     

Structural insights for the substrate recognition mechanism of LL-diaminopimelate aminotransferase

The enzymes involved in the lysine biosynthetic pathway have long been considered to be attractive targets for novel antibiotics due to the absence of this pathway in humans. Recently, a novel pyridoxal 5'-phosphate (PLP) dependent enzyme called LL-diaminopimelate aminotransferase (LL-DAP-AT) was identified in the lysine biosynthetic pathway of plants and Chlamydiae. Understanding its function and substrate recognition mechanism would be an important initial step toward designing novel antibiotics targeting LL-DAP-AT. The crystal structures of LL-DAP-AT from Arabidopsis thaliana in complex with various substrates and analogues have been solved recently. These structures revealed how L-glutamate and LL-DAP are recognized by LL-DAP-AT without significant conformational changes in the enzyme's backbone structure. This review article summarizes the recent developments in the structural characterization and the inhibitor design of LL-DAP-AT from A. thaliana. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

D-GLYCERATE 3-KINASE, the last unknown enzyme in the photorespiratory cycle in Arabidopsis, belongs to a novel kinase family

D-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C2 cycle, an indispensable ancillary metabolic pathway to the photosynthetic C3 cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C2 cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO2, providing direct evidence of the obligatory nature of the ultimate step of the C2 cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.     

L-Ascorbate biosynthesis in higher plants: the role of VTC2

In the past year, the last missing enzyme of the L-galactose pathway, the linear form of which appears to represent the major biosynthetic route to L-ascorbate (vitamin C) in higher plants, has been identified as a GDP-L-galactose phosphorylase. This enzyme catalyzes the first committed step in the synthesis of that vital antioxidant and enzyme cofactor. Here, we discuss how GDP-L-galactose phosphorylase enzymes, encoded in Arabidopsis by the paralogous VTC2 and VTC5 genes, function in concert with the other enzymes of the L-galactose pathway to provide plants with the appropriate levels of L-ascorbate. We hypothesize that regulation of L-ascorbate biosynthesis might occur at more than one step and warrants further investigation to allow for the manipulation of vitamin C levels in plants.     

Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme

In plants, a proposed ascorbate (vitamin C) biosynthesis pathway occurs via GDP-D-mannose (GDP-D-Man), GDP-L-galactose (GDP-L-Gal), and L-galactose. However, the steps involved in the synthesis of L-Gal from GDP-L-Gal in planta are not fully characterized. Here we present evidence for an in vivo role for L-Gal-1-P phosphatase in plant ascorbate biosynthesis. We have characterized a low ascorbate mutant (vtc4-1) of Arabidopsis thaliana, which exhibits decreased ascorbate biosynthesis. Genetic mapping and sequencing of the VTC4 locus identified a mutation (P92L) in a gene with predicted L-Gal-1-P phosphatase activity (At3g02870). Pro-92 is within a beta-bulge that is conserved in related myo-inositol monophosphatases. The mutation is predicted to disrupt the positioning of catalytic amino acid residues within the active site. Accordingly, L-Gal-1-P phosphatase activity in vtc4-1 was approximately 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-(3)H]Man into L-galactosyl residues suggesting that the mutation increases the availability of GDP-L-Gal for polysaccharide synthesis. Finally, a homozygous T-DNA insertion line, which lacks a functional At3g02870 gene product, is also ascorbate-deficient (50% of wild type) and deficient in L-Gal-1-P phosphatase activity. Genetic complementation tests revealed that the insertion mutant and VTC4-1 are alleles of the same genetic locus. The significantly lower ascorbate and perturbed L-Gal metabolism in vtc4-1 and the T-DNA insertion mutant indicate that L-Gal-1-P phosphatase plays a role in plant ascorbate biosynthesis. The presence of ascorbate in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to ascorbate biosynthesis.     

Molecular characterization of plastidic phosphoserine aminotransferase in serine biosynthesis from Arabidopsis

Serine biosynthesis in plants proceeds by two pathways; a photorespiratory pathway which is associated with photorespiration and a pathway from phosphoglycerate. A cDNA encoding plastidic phosphoserine aminotransferase (PSAT) which catalyzes the formation of phosphoserine from phosphohydroxypyruvate has been isolated from Arabidopsis thaliana . Genomic DNA blot analysis indicated that this enzyme is most probably encoded by a single gene and is mapped on the lower arm of chromosome 4. The deduced protein contains an N-terminal extension exhibiting the general features of a plastidic transit peptide, which was confirmed by subcellular organelle localization using GFP (green flourescence protein). Northern analysis indicated preferential expression of PSAT in roots of light-grown plants, supporting the idea that the phosphorylated pathway may play an important role in supplying the serine requirement of plants in non-green tissues. In situ hybridization analysis of PSAT revealed that the gene is generally expressed in all types of cells with a significantly higher amount in the meristem tissue of root tips.     

Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis

A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5- to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I(50) approximately 0.2 microm), whereas the mutant enzyme R264K was not significantly inhibited by 200 microm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants.     

Early steps of deoxyxylulose phosphate pathway in chromoplasts of higher plants

1-Deoxy-D-xylulose 5-phosphate and 2C-methyl-D-erythritol 4-phosphate have been shown as intermediates of the deoxyxylulose phosphate pathway used for terpenoid biosynthesis in plants and many microorganisms. In plants this non-mevalonate pathway is located in plastids. In order to investigate the formation of five carbon intermediates, chromoplasts from Capsicum annuum and Narcissus pseudonarcissus were incubated with isotope-labeled 1-deoxy-D-xylulose 5-phosphate or 2C-methyl-D-erythritol 4-phosphate. The downstream metabolites were detected and separated by reversed-phase ion-pair radio-HPLC and their structures elucidated by mass spectroscopy. Here we report the isolation and structural identification of 4-diphosphocytidyl-2C-methyl-D-erythritol and 2C-methyl-D-erythritol 2,4-cyclodiphosphate from chromoplasts; the genes of the corresponding enzymes had been previously identified from Escherichia coli and Arabidopsis.     

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1H,3H) pyrimidinedione 5′‐phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K_m values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.     

Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl-ACP glycerol-3- phosphate acyltransferase

The biosynthesis of phosphatidylglycerol represents a central pathway in lipid metabolism in all organisms. The enzyme catalyzing the first reaction of the pathway in the plastid, glycerol-3-phosphate acyl-acyl carrier protein acyltransferase, is thought to be encoded in Arabidopsis by the ATS1 locus. A number of genetic mutants deficient in this activity have been described. However, the corresponding mutant alleles have not yet been analyzed at the molecular level and a causal relationship between the mutant phenotypes and a deficiency at the ATS1 locus has not been established. The presence in all known ats1 mutants of near wild-type amounts of phosphatidylglycerol raised the question of whether an alternative pathway of phosphatidylglycerol assembly in the plastid exists. However, detailed analysis of several independent ats1 mutant alleles revealed that all are leaky. Reduction by RNAi of ats1-1 RNA levels in the ats1-1 mutant background led to a more severe growth phenotype (small green plants and reduced seed set), but did not decrease the relative amount of phosphatidylglycerol. In contrast, when the amount of ATS2 mRNA encoding the plastidic lysophosphatidic acid acyltransferase catalyzing the second reaction of the pathway was reduced by RNAi in the ats1-1 mutant background, phosphatidylglycerol amounts decreased, leading to a growth phenotype (small pale-yellow plants) that is reminiscent of the pgp1-1 mutant deficient in a late step of plastidic phosphatidylglycerol biosynthesis. These observations indicate coordinated regulation of plastid lipid metabolism and plant development.     

Organisation of the pantothenate (vitamin B5) biosynthesis pathway in higher plants

Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzyme A and acyl carrier protein, and is synthesised in Escherichia coli by four enzymic reactions. Ketopantoate hydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the first and last steps, respectively. Two genes encoding KPHMT and one for PtS were identified in the Arabidopsis thaliana genome, and cDNAs for all three genes were amplified by PCR. The cDNAs were able to complement their respective E. coli auxotrophs, demonstrating that they encoded functional enzymes. Subcellular localisation of the proteins was investigated using green fluorescent protein (GFP) fusions and confocal microscopy. The two KPHMT-GFP fusion proteins were targeted exclusively to mitochondria, whereas PtS-GFP was found in the cytosol. This implies that there must be transporters for pathway intermediates. KPHMT enzyme activity could be measured in purified mitochondria from both pea leaves and Arabidopsis suspension cultures. We investigated whether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes from E. coli, l-aspartate-alpha-decarboxylase (ADC) and ketopantoate reductase (KPR). No homologue of ADC could be identified using either conventional blast or searches with the program fugue in which the structure of the E. coli ADC was compared to all the annotated proteins in Arabidopsis. ADC also appears to be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria. In contrast, a putative Arabidopsis oxidoreductase with some similarity to KPR was identified with fugue.     

Isolating the Arabidopsis thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants. I. 5'-Phosphoribosyl-5-aminoimidazole synthetase.

We have initiated an investigation of the de novo purine nucleotide biosynthetic pathway in the plant Arabidopsis thaliana. Functional suppression of Escherichia coli auxotrophs allowed the direct isolation of expressed Arabidopsis leaf cDNAs. Using this approach we have successfully suppressed mutants in 4 of the 12 genes in this pathway. One of these cDNA clones, encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase (PUR5) has been characterized in detail. Analysis of genomic DNA suggests that the Arabidopsis genome contains a single AIR synthetase gene. Analysis of the cDNA sequence and mRNA size suggests that this enzyme activity is encoded by a monofunctional polypeptide, similar to that of bacteria and unlike other eukaryotes. The Arabidopsis AIR synthetase contains a basic hydrophobic transit peptide consistent with transport into chloroplasts. Comparison of both the predicted amino acid and nucleotide sequence from Arabidopsis to those of eight other distant organisms suggests that the plant sequence is more similar to the bacterial sequences than to other eukaryotic sequences. This study provides the groundwork for future investigations into the regulation of de novo purine biosynthesis in plants. Additionally, we have demonstrated that functional suppression of bacterial mutants may provide a useful method for cloning a variety of plant genes.