Histidine biosynthesis in plants.

The study of histidine metabolism has never been at the forefront of interest in plant systems despite the significant role that the analysis of this pathway has played in development of the field of molecular genetics in microbes. With the advent of methods to analyze plant gene function by complementation of microbial auxotrophic mutants and the complete analysis of plant genome sequences, strides have been made in deciphering the histidine pathway in plants. The studies point to a complex evolutionary origin of genes for histidine biosynthesis. Gene regulation studies have indicated novel regulatory networks involving histidine. In addition, physiological studies have indicated novel functions for histidine in plants as chelators and transporters of metal ions. Recent investigations have revealed intriguing connections of histidine in plant reproduction. The exciting new information suggests that the study of plant histidine biosynthesis has finally begun to flower.

Regulation of the plant-type 5'-adenylyl sulfate reductase by oxidative stress.

5'-Adenylyl sulfate (APS) reductase (EC 1.8.4.9) catalyzes a key reaction in the plant sulfate assimilation pathway leading to the synthesis of cysteine and the antioxidant glutathione. In Arabidopsis thaliana APS reductase is encoded by a family of three genes. In vitro biochemical studies revealed that the enzyme product derived from one of them (APR1) is activated by oxidation, probably through the formation of a disulfide bond. The APR1 enzyme is 45-fold more active when expressed in a trxB strain of Escherichia coli than in a trxB(+) wild type. The enzyme is inactivated in vitro by treatment with disulfide reductants and is reactivated with thiol oxidants. Redox titrations show that the regulation site has a midpoint potential of -330 mV at pH 8.5 and involves a two-electron redox reaction. Exposure of a variety of plants to ozone induces a rapid increase in APS reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. During the response to ozone, the level of immunodetectable APS reductase enzyme does not increase. Treatment of A. thaliana seedlings with oxidized glutathione or paraquat induces APS reductase activity even when transcription or translation is blocked with inhibitors. The results suggest that a posttranslational mechanism controls APS reductase. A model is proposed whereby redox regulation of APS reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress.

Recombinant Arabidopsis SQD1 converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro.

The sulfolipid sulfoquinovosyldiacylglycerol is a component of plant photosynthetic membranes and represents one of the few naturally occurring sulfonic acids with detergent properties. Sulfolipid biosynthesis involves the transfer of sulfoquinovose, a 6-deoxy-6-sulfoglucose, from UDP-sulfoquinovose to diacylglycerol. The formation of the sulfonic acid precursor, UDP-sulfoquinovose, from UDP-glucose and a sulfur donor is proposed to be catalyzed by the bacterial SQDB proteins or the orthologous plant SQD1 proteins. To investigate the underlying enzymatic mechanism and to elucidate the de novo synthesis of sulfonic acids in biological systems, we developed an in vitro assay for the recombinant SQD1 protein from Arabidopsis thaliana. Among different possible sulfur donors tested, sulfite led to the formation of UDP-sulfoquinovose in the presence of UDP-glucose and SQD1. An SQD1 T145A mutant showed greatly reduced activity. The UDP-sulfoquinovose formed in this assay was identified by co-chromatography with standards and served as substrate for the sulfolipid synthase associated with spinach chloroplast membranes. Approximate K(m) values of 150 microm for UDP-glucose and 10 microm for sulfite were established for SQD1. Based on our results, we propose that SQD1 catalyzes the formation of UDP-sulfoquinovose from UDP-glucose and sulfite, derived from the sulfate reduction pathway in the chloroplast.

Differential subcellular localization and expression of ATP sulfurylase and 5'-adenylylsulfate reductase during ontogenesis of Arabidopsis leaves indicates that cytosolic and plastid forms of ATP sulfurylase may have specialized functions.

ATP sulfurylase and 5'-adenylylsulfate (APS) reductase catalyze two reactions in the sulfate assimilation pathway. Cell fractionation of Arabidopsis leaves revealed that ATP sulfurylase isoenzymes exist in the chloroplast and the cytosol, whereas APS reductase is localized exclusively in chloroplasts. During development of Arabidopsis plants the total activity of ATP sulfurylase and APS reductase declines by 3-fold in leaves. The decline in APS reductase can be attributed to a reduction of enzyme during aging of individual leaves, the highest activity occurring in the youngest leaves and the lowest in fully expanded leaves. By contrast, total ATP sulfurylase activity declines proportionally in all the leaves. The distinct behavior of ATP sulfurylase can be attributed to reciprocal expression of the chloroplast and cytosolic isoenzymes. The chloroplast form, representing the more abundant isoenzyme, declines in parallel with APS reductase during aging; however, the cytosolic form increases over the same period. In total, the results suggest that cytosolic ATP sulfurylase plays a specialized function that is probably unrelated to sulfate reduction. A plausible function could be in generating APS for sulfation reactions.

Characterization of sulfate assimilation in marine algae focusing on the enzyme 5'-adenylylsulfate reductase.

5'-Adenylylsulfate (APS) reductase was characterized in diverse marine algae. A cDNA encoding APS reductase from Enteromorpha intestinalis (EAPR) was cloned by functional complementation of an Escherichia coli cysH mutant. The deduced amino acid sequence shows high homology with APS reductase (APR) from flowering plants. Based on the probable transit peptide cleavage site the mature protein is 45.7 kD. EAPR expressed as a His-tagged recombinant protein catalyzes reduced glutathione-dependent reduction of APS to sulfite, exhibiting a specific activity of approximately 40 micromol min(-1) mg protein(-1) and Michealis-Menten kinetic constants of approximately 1.4 mM for reduced glutathione and approximately 6.5 microM for APS. APR activity and expression were studied in relation to the production of 3-dimethylsulfoniopropionate (DMSP), a sulfonium compound produced by many marine algae. A diverse group of DMSP-producing species showed extremely high enzyme activity (up to 400 times that found in flowering plants). Antibodies raised against a conserved peptide of APR strongly cross-reacted with a protein of 45 kD in several chlorophytes but insignificantly with chromophytes. In the chlorophyte Tetraselmis sp., APR activity varies significantly during the culture cycle and does not follow the changes in cellular DMSP content. However, a positive correlation was found between cell-based APR activity and specific growth rate.

Functional characterization of a gene encoding a fourth ATP sulfurylase isoform from Arabidopsis thaliana.

ATP sulfurylase (ATP: sulfate adenylyl transferase, EC 2.7.7.4), the first enzyme of the sulfate assimilation pathway, is present in the chloroplast and cytosol of plants. In Arabidopsis thaliana cDNA cloning revealed the existence of three ATP sulfurylase isoforms (APS1, -2, and -3) all of which appear to be localized in plastids. In the present study the cytosolic isoform was sought by searching the expressed sequence tag (EST) database and by screening A. thaliana genomic libraries. A fourth isoform, APS4, was identified, but it also encodes a plastid-localized isoform. The APS genes all contain four introns. The introns are located at identical positions within the coding sequence of each of the APS genes. A putative TATA box was identified in the promoter of the APS3 and APS4 genes, but no regions of sequence similarity were found among the other promoters. Combined analysis of an APS4 cDNA and genomic clone revealed that the deduced protein is 469 amino acids and is most homologous to the A. thaliana APS1 subclass. The APS4 cDNA was able to functionally complement a yeast ATP sulfurylase (met3) mutant and the recombinant enzyme displayed ATP sulfurylase activity. The APS4 protein exhibits a plastid targeting peptide at its amino terminus that, when fused to green fluorescent protein, was able to target the reporter to chloroplasts. APS4 mRNA was detected at a similar steady-state level in roots and leaves, and its expression was not induced by sulfur starvation or by O-acetylserine treatment. Having identified a fourth plastid-localized ATP sulfurylase, the origin of cytosolic isoform in A. thaliana remains unclear. Based on sequence analysis, it is hypothesized that APS2 may encode the cytosolic ATP sulfurylase.

Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria.

A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase. The B. cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E. coli CysH. The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase. It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product. Insertional knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified P. aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 micromol. min(-1). mg of protein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.

Identification of the gene encoding homoserine kinase from Arabidopsis thaliana and characterization of the recombinant enzyme derived from the gene.

Homoserine kinase (EC 2.7.1.39) catalyzes the formation of O-phospho-l-homoserine, a branch point intermediate in the pathways for Met and Thr in plants. A genomic open reading frame located on the top arm of chromosome II and a corresponding cDNA have been identified from Arabidopsis thaliana that encode homoserine kinase. The HSK gene is composed of an 1113-bp continuous open reading frame that could produce a 38-kDa protein. The gene product has homology with homoserine kinase from bacteria and fungi. It contains a conserved motif, known as GHMP, found in a group of ATP-dependent metabolite kinases and thought to comprise the ATP binding site. The amino-terminal 50 amino acids of the HSK protein show features of a transit peptide for localization to plastids. Genomic blot analysis revealed that there is a single locus in A. thaliana to which the HSK cDNA hybridizes. The HSK protein expressed as a His-tagged construct in Escherichia coli shows a specific activity in an l-homoserine-dependent ADP synthesis assay of 3.09 +/- 0.25 micromol min(-1) mg(-1) protein at pH 8.5 and 37 degrees C. The apparent K(m) values are 0.40 mM for l-homoserine and 0.32 mM for Mg-ATP. Other hydroxylated compounds are not used as substrates. The enzyme requires 40 mM K(+) and 3 mM Mg(2+) for activity. It has an unusually high temperature optimum, yet it is very unstable, losing more than 80% of its activity after a single cycle of freeze-thawing. The HSK enzyme shows no significant regulation by amino acids in vitro.

Evidence for autoregulation of cystathionine gamma-synthase mRNA stability in Arabidopsis.

Control of messenger RNA (mRNA) stability serves as an important mechanism for regulating gene expression. Analysis of Arabidopsis mutants that overaccumulate soluble methionine (Met) revealed that the gene for cystathionine gamma-synthase (CGS), the key enzyme in Met biosynthesis, is regulated at the level of mRNA stability. Transfection experiments with wild-type and mutant forms of the CGS gene suggest that an amino acid sequence encoded by the first exon of CGS acts in cis to destabilize its own mRNA in a process that is activated by Met or one of its metabolites.

Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem-translocated compound.

Sulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO4(2-). Similar responses occurred in split-root experiments where only a portion of the root system was S-deprived, suggesting that the regulation involves inter-organ signaling. Phloem-translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO4(2-) uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split-root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO4(2-) restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO4(2-) transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.

The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis.

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.

Overexpression of ATP sulfurylase in indian mustard leads to increased selenate uptake, reduction, and tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1. 5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.

Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5'-adenylylsulfate reductase.

5'-Adenylylsulfate (APS) reductase (EC 1.8.99.-) catalyzes the reduction of activated sulfate to sulfite in plants. The evidence presented here shows that a domain of the enzyme is a glutathione (GSH)-dependent reductase that functions similarly to the redox cofactor glutaredoxin. The APR1 cDNA encoding APS reductase from Arabidopsis thaliana is able to complement the cysteine auxotrophy of an Escherichia coli cysH [3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase] mutant, only if the E. coli strain produces glutathione. The purified recombinant enzyme (APR1p) can use GSH efficiently as a hydrogen donor in vitro, showing aKm[GSH] approximately of 0.6 mM. Gene dissection was used to express separately the regions of APR1p from amino acids 73-327 (the R domain), homologous with microbial PAPS reductase, and from amino acids 328-465 (the C domain), homologous with thioredoxin. The R and C domains alone are inactive in APS reduction, but the activity is partially restored by mixing the two domains. The C domain shows a number of activities that are typical of E. coli glutaredoxin rather than thioredoxin. Both the C domain and APR1p are highly active in GSH-dependent reduction of hydroxyethyldisulfide, cystine, and dehydroascorbate, showing a Km[GSH] in these assays of approximately 1 mM. The R domain does not show these activities. The C domain is active in GSH-dependent reduction of insulin disulfides and ribonucleotide reductase, whereas APR1p and R domain are inactive. The C domain can substitute for glutaredoxin in vivo as demonstrated by complementation of an E. coli mutant, underscoring the functional similarity between the two enzymes.

APS kinase from Arabidopsis thaliana: genomic organization, expression, and kinetic analysis of the recombinant enzyme.

The gene encoding 5'-adenylylsulfate (APS) kinase (EC 2.7.1.25) (APK) was cloned from Arabidopsis thaliana. There is a single APK locus in A. thaliana. The coding sequence of the gene is composed of 7 exons, interrupted by 6 introns. A transcriptional initiation site was detected 120 bp 5' of the initiation codon. APK mRNA is slightly more abundant in leaves than in roots of A. thaliana and its level does not change in response to sulfur starvation. The APK protein, synthesized in vitro, is able to enter isolated intact chloroplasts. Recombinant APS kinase shows maximal activity at 10 microM APS with 5 mM ATP, but it is inhibited at APS concentrations above 10 microM. The inhibition is alleviated at higher ATP concentrations. Reciprocal plot analysis showed that the theoretical Vmax is approximately 1.2 mumol min-1 mg-1 at 25 degrees C, pH 8.0; the K(m) values are 3.6 microM APS and 1.8 mM ATP.

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana.

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.

Cloning and bacterial expression of adenosine-5'-triphosphate sulfurylase from the enteric protozoan parasite Entamoeba histolytica.

A gene encoding adenosine-5'-triphosphate sulfurylase (AS) was cloned from the enteric protozoan parasite Entamoeba histolytica by polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the protein from a variety of organisms. The deduced amino acid sequence of E. histolytica AS revealed a calculated molecular mass of 47925 Da and an unusual basic pI of 9.38. The amebic protein sequence showed 23-48% identities with AS from bacteria, yeasts, fungi, plants, and animals with the highest identities being to Synechocystis sp. and Bacillus subtilis (48 and 44%, respectively). Four conserved blocks including putative sulfate-binding and phosphate-binding regions were highly conserved in the E. histolytica AS. The upstream region of the AS gene contained three conserved elements reported for other E. histolytica genes. A recombinant E. histolytica AS revealed enzymatic activity, measured in both the forward and reverse directions. Expression of the E. histolytica AS complemented cysteine auxotrophy of the AS-deficient Escherichia coli strains. Genomic hybridization revealed that the AS gene exists as a single copy gene. In the literature, this is the first description of an AS gene in Protozoa.

Plant sulfur metabolism--the reduction of sulfate to sulfite.

Until recently the pathway by which plants reduce activated sulfate to sulfite was unresolved. Recent findings on two enzymes termed 5'-adenylylsulfate (APS) sulfotransferase and APS reductase have provided new information on this topic. On the basis of their similarities it is now proposed that these proteins are the same enzyme. These discoveries confirm that the sulfate assimilation pathway in plants differs from that in other sulfate assimilating organisms.

Regulation of sulfur assimilation in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana.

Proton/sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants. Here we report the cloning and characterization of an Arabidopsis thaliana gene, AST68, a new member of the sulfate transporter gene family in higher plants. Sequence analysis of cDNA and genomic clones of AST68 revealed that the AST68 gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a Saccharomyces cerevisiae mutant lacking a sulfate transporter gene. Southern hybridization and restriction fragment length polymorphism mapping confirmed that AST68 is a single-copy gene that maps to the top arm of chromosome 5. Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of AST68 increased specifically in roots up to 9-fold by sulfate starvation. In situ hybridization experiments revealed that AST68 transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis. Among all the structural genes for sulfate assimilation, sulfate transporter (AST68), APS reductase (APR1), and serine acetyltransferase (SAT1) were inducible by sulfate starvation in A. thaliana. The sulfate transporter (AST68) exhibited the most intensive and specific response in roots, indicating that AST68 plays a central role in the regulation of sulfate assimilation in plants.

A new route for synthesis of dimethylsulphoniopropionate in marine algae.

The 3-dimethylsulphoniopropionate (DMSP) produced by marine algae is the main biogenic precursor of atmospheric dimethylsulphide (DMS). This biogenic DMS, formed by bacterial and algal degradation of DMSP, contributes about 1.5 x 10(13) g of sulphur to the atmosphere annually, and plays a major part in the global sulphur cycle, in cloud formation and potentially in climate regulation. Although DMSP biosynthesis has been partially elucidated in a higher plant, nothing is known about how algae make DMSP except that the whole molecule is derived from methionine. Here we use in vivo isotope labelling to demonstrate that DMSP synthesis in the green macroalga Enteromorpha intestinalis proceeds by a route entirely distinct from that in higher plants. From methionine, the steps are transamination, reduction and S-methylation to give the novel sulphonium compound 4-dimethylsulphonio-2-hydroxybutyrate (DMSHB), which is oxidatively decarboxylated to DMSP. The key intermediate DMSHB was also identified in three diverse phytoplankton species, indicating that the same pathway operates in other algal classes that are important sources of DMS. The fact that a transamination initiates this pathway could help explain how algal DMSP (and thereby DMS) production is enhanced by nitrogen deficiency.