Francisco J. Sandoval

Flavin Nucleotide Metabolism in Plants: MONOFUNCTIONAL ENZYMES SYNTHESIZE FAD IN PLASTIDS*

FAD synthetases (EC 2.7.7.2) catalyze biosynthesis of FAD from FMN and ATP. Monofunctional FAD synthetases are known to exist in mammals and yeast; bifunctional enzymes also catalyzing phosphorylation of riboflavin to FMN are known to exist in bacteria. Previously known eukaryotic enzymes with FAD synthetase activity have no sequence similarity to prokaryotic enzymes with riboflavin kinase and FAD synthetase activities. Proteins homologous to bacterial bifunctional FAD synthetases, yet shorter and lacking amino acid motifs at the C terminus, were found by bioinformatic analyses in vascular plant genomes, suggesting that plants contain a type of FAD synthetase previously known to exist only in prokaryotes. The Arabidopsis thaliana genome encodes two of such proteins. Both proteins, which we named AtRibF1 and AtRibF2, carry N-terminal extensions with characteristics of organellar targeting peptides. AtRibF1 and AtRibF2 cDNAs were cloned by reverse transcription-PCR. Only FAD synthetase activity was detected in the recombinant enzymes produced in Escherichia coli. FMN and ATP inhibited both enzymes. Kinetic parameters of AtRibF1 and AtRibF2 for the two substrates were similar. Confocal microscopy of protoplasts transformed with enhanced green fluorescence protein-fused proteins showed that AtRibF1 and AtRibF2 are targeted to plastids. In agreement with subcellular localization to plastids, Percoll-isolated chloroplasts from pea (Pisum sativum) synthesized FAD from imported riboflavin. Riboflavin kinase, FMN hydrolase, and FAD pyrophosphatase activities were detected in Percoll-isolated chloroplasts and mitochondria from pea. We propose from these new findings a model for subcellular distribution of enzymes that synthesize and hydrolyze flavin nucleotides in plants.

An FMN Hydrolase Is Fused to a Riboflavin Kinase Homolog in Plants

Riboflavin kinases catalyze synthesis of FMN from riboflavin and ATP. These enzymes have to date been cloned from bacteria, yeast, and mammals, but not from plants. Bioinformatic approaches suggested that diverse plant species, including many angiosperms, two gymnosperms, a moss (Physcomitrella patens), and a unicellular green alga (Chlamydomonas reinhardtii), encode proteins that are homologous to riboflavin kinases of yeast and mammals, but contain an N-terminal domain that belongs to the haloacid dehalogenase superfamily of enzymes. The Arabidopsis homolog of these proteins was cloned by RT-PCR, and was shown to have riboflavin kinase and FMN hydrolase activities by characterizing the recombinant enzyme produced in Escherichia coli. Both activities of the purified recombinant Arabidopsis enzyme (AtFMN/FHy) increased when the enzyme assays contained 0.02% Tween 20. The FMN hydrolase activity of AtFMN/FHy greatly decreased when EDTA replaced Mg2+ in the assays, as expected for a member of the Mg2+-dependent haloacid dehalogenase family. The functional overexpression of the individual domains in E. coli establishes that the riboflavin kinase and FMN hydrolase activities reside, respectively, in the C-terminal (AtFMN) and N-terminal (AtFHy) domains of AtFMN/FHy. Biochemical characterization of AtFMN/FHy, AtFMN, and AtFHy shows that the riboflavin kinase and FMN hydrolase domains of AtFMN/FHy can be physically separated, with little change in their kinetic properties.

Repression of Sulfate Assimilation Is an Adaptive Response of Yeast to the Oxidative Stress of Zinc Deficiency

The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. Previous studies identified over 80 genes activated by Zap1 in zinc-limited cells. In this report, we identified 36 genes repressed in a zinc- and Zap1-responsive manner. As a result, we have identified a new mechanism of Zap1-mediated gene repression whereby transcription of the MET3, MET14, and MET16 genes is repressed in zinc-limited cells. These genes encode the first three enzymes of the sulfate assimilation pathway. We found that MET30, encoding a component of the SCFMet30 ubiquitin ligase, is a direct Zap1 target gene. MET30 expression is increased in zinc-limited cells, and this leads to degradation of Met4, a transcription factor responsible for MET3, MET14, and MET16 expression. Thus, Zap1 is responsible for a decrease in sulfate assimilation in zinc-limited cells. We further show that cells that are unable to down-regulate sulfate assimilation under zinc deficiency experience increased oxidative stress. This increased oxidative stress is associated with an increase in the NADP+/NADPH ratio and may result from a decrease in NADPH-dependent antioxidant activities. These studies have led to new insights into how cells adapt to nutrient-limiting growth conditions.

One-carbon metabolism in plants: characterization of a plastid serine hydroxymethyltransferase

SHMT (serine hydroxymethyltransferase; EC 2.1.2.1) catalyses reversible hydroxymethyl group transfer from serine to H4PteGlun (tetrahydrofolate), yielding glycine and 5,10-methylenetetrahydrofolate. In plastids, SHMTs are thought to catalytically direct the hydroxymethyl moiety of serine into the metabolic network of H4PteGlun-bound one-carbon units. Genes encoding putative plastid SHMTs were found in the genomes of various plant species. SHMT activity was detected in chloroplasts in pea (Pisum sativum) and barley (Hordeum vulgare), suggesting that plastid SHMTs exist in all flowering plants. The Arabidopsis thaliana genome encodes one putative plastid SHMT (AtSHMT3). Its cDNA was cloned by reverse transcription-PCR and the encoded recombinant protein was produced in Escherichia coli. Evidence that AtSHMT3 is targeted to plastids was found by confocal microscopy of A. thaliana protoplasts transformed with proteins fused to enhanced green fluorescent protein. Characterization of recombinant AtSHMT3 revealed that substrate affinity for and the catalytic efficiency of H4PteGlu1-8 increase with n, and that H4PteGlu1-8 inhibit AtSHMT3. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate with one and five glutamate residues inhibited AtSHMT3-catalysed hydroxymethyl group transfer from serine to H4PteGlu6, with the pentaglutamylated inhibitors being more effective. Calculations revealed inhibition with 5-methyltetrahydrofolate or 5-formyltetrahydrofolate resulting in little reduction in AtSHMT3 activity under folate concentrations estimated for plastids.

Bacterial Attraction and Quorum Sensing Inhibition in Caenorhabditis elegans Exudates

Caenorhabditis elegans, a bacterivorous nematode, lives in complex rotting fruit, soil, and compost environments, and chemical interactions are required for mating, monitoring population density, recognition of food, avoidance of pathogenic microbes, and other essential ecological functions. Despite being one of the best-studied model organisms in biology, relatively little is known about the signals that C. elegans uses to interact chemically with its environment or as defense. C. elegans exudates were analyzed by using several analytical methods and found to contain 36 common metabolites that include organic acids, amino acids, and sugars, all in relatively high abundance. Furthermore, the concentrations of amino acids in the exudates were dependent on developmental stage. The C. elegans exudates were tested for bacterial chemotaxis using Pseudomonas putida (KT2440), a plant growth promoting rhizobacterium, Pseudomonas aeruginosa (PAO1), a soil bacterium pathogenic to C. elegans, and Escherichia coli (OP50), a non-motile bacterium tested as a control. The C. elegans exudates attracted the two Pseudomonas species, but had no detectable antibacterial activity against P. aeruginosa. To our surprise, the exudates of young adult and adult life stages of C. elegans exudates inhibited quorum sensing in the reporter system based on the LuxR bacterial quorum sensing (QS) system, which regulates bacterial virulence and other factors in Vibrio fischeri. We were able to fractionate the QS inhibition and bacterial chemotaxis activities, thus demonstrating that these activities are chemically distinct. Our results demonstrate that C. elegans can attract its bacterial food and has the potential of partially regulating the virulence of bacterial pathogens by inhibiting specific QS systems.

Nuclear Localised MORE SULPHUR ACCUMULATION1 Epigenetically Regulates Sulphur Homeostasis in Arabidopsis thaliana

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S-adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over-accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation.

An FMN Hydrolase of the Haloacid Dehalogenase Superfamily Is Active in Plant Chloroplasts

Background: Flavin nucleotide metabolism in plants is incompletely understood. Results: Gene At1g79790 encodes an FMN-specific hydrolase (AtcpFHy1) that localizes to plastids. Conclusion: This is the first study reporting cloning and characterization of an FMN hydrolase from plastids. Significance: This study advances the knowledge of flavin nucleotide metabolism in plants. FMN hydrolases catalyze dephosphorylation of FMN to riboflavin. Although these enzymes have been described in many organisms, few had their corresponding genes cloned and their recombinant proteins biochemically characterized, and none had their physiological roles determined. We found previously that FMN hydrolase activity in pea chloroplasts is Mg2+-dependent, suggesting an enzyme of the haloacid dehalogenase (HAD) superfamily. In this study, a new FMN hydrolase was purified by multistep chromatography after ammonium sulfate precipitation. The molecular weight of the native protein was estimated at ∼59,400, a dimer of about twice the predicted molecular weight of most HAD superfamily phosphatases. After SDS-PAGE of the partially purified material, two separate protein bands within 25–30 kDa were extracted from the gel and analyzed by nanoLC-MS/MS. Peptide sequence matching to the protein samples suggested the presence of three HAD-like hydrolases. cDNAs for sequence homologs from Arabidopsis thaliana of these proteins were expressed in Escherichia coli. Activity screening of the encoded proteins showed that the At1g79790 gene encodes an FMN hydrolase (AtcpFHy1). Plastid localization of AtcpFHy1 was confirmed using fluorescence microscopy of A. thaliana protoplasts transiently expressing the N-terminal fusion of AtcpFHy1 to enhanced green fluorescent protein. Phosphatase activity of AtcpFHy1 is FMN-specific, as assayed with 19 potential substrates. Kinetic parameters and pH and temperature optima for AtcpFHy1 were determined. A phylogenetic analysis of putative phosphatases of the HAD superfamily suggested distinct evolutionary origins for the plastid AtcpFHy1 and the cytosolic FMN hydrolase characterized previously.

Folate polyglutamylation eliminates dependence of activity on enzyme concentration in mitochondrial serine hydroxymethyltransferases from Arabidopsis thaliana

The reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT) is the major one-carbon unit source for essential metabolic processes. The Arabidopsis thaliana genome encodes seven SHMT isozymes localized in mitochondria, plastids, nuclei, and the cytosol. Knowledge of the biochemical properties of each isozyme is central to understanding and manipulating one-carbon metabolism in plants. We heterologously expressed and purified three recombinant SHMTs from A. thaliana (AtSHMTs) putatively localized in mitochondria (two) and the cytosol (one). Their biochemical properties were characterized with respect to the impact of folate polyglutamylation on substrate saturation kinetics. The two mitochondrial AtSHMTs, but not the cytosolic one, had increased turnover rates at higher (>0.4ng/μL) enzyme concentrations in the presence of monoglutamylated folate substrates, but not in the presence of pentaglutamylated folate substrates. We found no experimental support for a change in oligomerization state over the range of enzyme concentration studied. Modeling of the enzyme structures presented features that may explain the activity differences between the mitochondrial and cytosolic isozymes.