O-acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana

The synthesis of cysteine is positioned at a decisive stage of assimilatory sulphate reduction, marking the fixation of inorganic sulphide into a carbon skeleton. O-acetylserine (thiol) lyase (OAS-TL) catalyses the reaction of inorganic sulphide with O-acetylserine (OAS). Despite its prominent position in the pathway OAS-TL is generally regarded as a non-limiting enzyme without regulatory function, due to low substrate affinities and semi-constitutive expression patterns. To resolve this apparent contradiction, the kinetic properties of three OAS-TLs from Arabidopsis thaliana, localized in the cytosol (A), plastids (B), and mitochondria (C), were analysed. The recombinant expressed OAS-TLs were purified to apparent homogeneity without any fusion tag to maintain their native forms. The proteins displayed high specific activities of 550-900 micromol min(-1) mg(-1). Using an improved and highly sensitive assay method for cysteine determination, the apparent K(m)(sulphide) was 3-6 microM for OAS-TL A, B, and C and thus 10-100 times lower than previously reported for plant OAS-TLs. K(m)(OAS) was between 310 microM and 690 microM for OAS-TL isoform A, B, and C, whereas the apparent dissociation binding constant for OAS was much lower (K(d)<1 microM OAS). A HPLC method was developed for OAS quantification that revealed fast increases of the cellular OAS concentration in response to sulphate deprivation. The observed fluctuations of intracellular OAS concentrations, combined with the OAS dissociation constant and the catalytic properties of OAS-TL, support the model of a dynamic cysteine synthesis system with regulatory function as can be expected from the position of the reaction in the sulphur assimilation pathway.     

Analysis of the Arabidopsis O-acetylserine(thiol)lyase gene family demonstrates compartment-specific differences in the regulation of cysteine synthesis

Cys synthesis in plants takes place in plastids, cytosol, and mitochondria. Why Cys synthesis is required in all compartments with autonomous protein biosynthesis and whether Cys is exchanged between them has remained enigmatic. This question was addressed using Arabidopsis thaliana T-DNA insertion lines deficient in the final step of Cys biosynthesis catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL). Null alleles of oastlA or oastlB alone showed that cytosolic OAS-TL A and plastid OAS-TL B were completely dispensable, although together they contributed 95% of total OAS-TL activity. An oastlAB double mutant, relying solely on mitochondrial OAS-TL C for Cys synthesis, showed 25% growth retardation. Although OAS-TL C alone was sufficient for full development, oastlC plants also showed retarded growth. Targeted affinity purification identified the major OAS-TL-like proteins. Two-dimensional gel electrophoresis and mass spectrometry showed no compensatory changes of OAS-TL isoforms in the four mutants. Steady state concentrations of Cys and glutathione and pulse-chase labeling with [35S]sulfate indicated strong perturbation of primary sulfur metabolism. These data demonstrate that Cys and also sulfide must be sufficiently exchangeable between cytosol and organelles. Despite partial redundancy, the mitochondria and not the plastids play the most important role for Cys synthesis in Arabidopsis.     

Molecular basis of cysteine biosynthesis in plants: structural and functional analysis of O-acetylserine sulfhydrylase from Arabidopsis thaliana

In plants, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment and provides the only metabolic sulfide donor for the generation of methionine, glutathione, phytochelatins, iron-sulfur clusters, vitamin cofactors, and multiple secondary metabolites. O-Acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-acetylserine into cysteine. Here we describe the 2.2 A resolution crystal structure of OASS from Arabidopsis thaliana (AtOASS) and the 2.7 A resolution structure of the AtOASS K46A mutant with PLP and methionine covalently linked as an external aldimine in the active site. Although the plant and bacterial OASS share a conserved set of amino acids for PLP binding, the structure of AtOASS reveals a difference from the bacterial enzyme in the positioning of an active site loop formed by residues 74-78 when methionine is bound. Site-directed mutagenesis, kinetic analysis, and ligand binding titrations probed the functional roles of active site residues. These experiments indicate that Asn(77) and Gln(147) are key amino acids for O-acetylserine binding and that Thr(74) and Ser(75) are involved in sulfur incorporation into cysteine. In addition, examination of the AtOASS structure and nearly 300 plant and bacterial OASS sequences suggest that the highly conserved beta8A-beta9A surface loop may be important for interaction with serine acetyltransferase, the other enzyme in cysteine biosynthesis. Initial protein-protein interaction experiments using AtOASS mutants targeted to this loop support this hypothesis.     

Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases,enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana

A 13.8 kb DNA sequence containing the promoters and the structural genes of the Arabidopsis thaliana nit2/nit1/nit3 gene cluster has been isolated and characterized. The coding regions of nit2, nit1 and nit3 spanned 1.9, 1.8 and 2.1 kb, respectively. The architecture of the three genes is highly conserved. Each isoform consists of five exons separated by four introns. The introns are very similar with respect to size and position, but differ considerably in sequence composition. In contrast to the coding sequences the three promoters are very different in sequence, size and in their repertoire of cis elements, suggesting differential regulation of the three nitrilase isoenzymes by the developmental program of the plant and by diverse environmental factors. The nit1 promoter was subjected to analysis in planta. Translational fusions placing the nit1 full-length promoter and a series of 5-deletion fragments in front of the uidA gene encoding -glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum. GUS expression was highest in fully expanded leaves and in the shoot apex as well as in the apices of developing lateral buds, whereas the GUS activity displayed by developing younger leaflets was restricted to the tips of the expanding leaves. Within the root tissue GUS expression was restricted to the root tips and the tips of newly forming lateral roots. Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues.     

The glycosyltransferase gene encoding the enzyme catalyzing the first step of mycothiol biosynthesis (mshA)

Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins). A transposon mutant of Mycobacterium smegmatis shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated mshA. The ortholog in Mycobacterium tuberculosis, Rv0486, was used to complement the mutant phenotype.     

Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol)-lyase].

Cysteine synthase [O-acetyl-L-serine(thiol)-lyase, EC 4.2.99.8] (CSase), which is responsible for the terminal step of cysteine biosynthesis, catalyzes the formation of L-cysteine from O-acetyl-L-serine (OAS) and hydrogen sulfide. Three T-DNA vectors carrying a spinach (Spinacia oleracea) cytoplasmic CSase A cDNA (K. Saito, N. Miura, M. Yamazaki, H. Horano, I. Murakoshi [1992] Proc Natl Acad Sci USA 89: 8078-8082) were constructed as follows: pCSK3F, cDNA driven by the cauliflower mosaic virus (CaMV) 35S RNA promoter with a sense orientation; pCSK3R, cDNA driven by the CaMV 355 promoter with an antisense orientation; pCSK4F, cDNA fused with the sequence for chloroplast-targeting transit peptide of pea ribulose-1,5-biphosphate carboxylase small subunit driven by the CaMV 35S promoter with a sense orientation. These chimeric genes were transferred into tobacco (Nicotiana tabacum) with Agrobacterium-mediated transformation, and self-fertilized progeny were obtained. CSase activities in cell-free extracts of pCSK3F and pCSK4F transformants were 2- to 3-fold higher than those of control and pCSK3R plants. CSase activities in chloroplasts of pCSK4F transformants were severalfold higher than those of control and pCSK3F plants, indicating that the foreign CSase protein is transported and accumulated in a functionally active form in chloroplasts of pCSK4F plants. Isolated chloroplasts of a pCSK4F transformant had a more pronounced ability to form cysteine in response to addition of OAS and sulfur compounds than those of a control plant. In particular, feeding of OAS and sulfite resulted in enhanced cysteine formation, which required photoreduction of sulfite in chloroplasts. The enhanced cysteine formation in a pCSK4F plant responding to sulfite was also observed in leaf discs. In addition, these leaf discs were partially resistant to sulfite toxicity, possibly due to metabolic detoxification of sulfite by fixing into cysteine. These results suggested that overaccumulated foreign CSase in chloroplasts could modulate biosynthetic flow of cysteine in response to sulfur stress.     

Cloning and functional characterization of an O-acetylserine(thiol)lyase-encoding gene in wild soybean (Glycine soja)

The terminal step of soybean cysteine synthesis is catalyzed by O-acetylserine(thiol)lyase (OAS-TL, EC 2.5.1.47). In this study, we isolated and characterized an OAS-TL gene from a wild soybean material (designated as GsOAS-TL1). GsOAS-TL1 cDNA sequence showed strict conservation at both nucleotide and amino acid levels compared with that from cultivated soybean. Genomic structure analysis of GsOAS-TL1 indicated that it contained 10 exons and 9 introns in the coding region with conserved exon sizes and intron locations compared with Arabidopsis thaliana OAS-TL-like genes. Among the complete GsOAS-TL1 cDNA and three part-deletion fragments, only expression of the full-length cDNA could rescue the NK3 cys⁻ Escherichia coli auxotroph, which was coherent with the assayed enzyme activity of purified fusion proteins. For RT-PCR analysis in different wild soybean tissues, GsOAS-TL1 showed lower expression in roots and developing seeds, whereas total OAS-TL activity of corresponding tissues showed significantly higher level in seeds than other tissues. To our knowledge, this is the first report on cloning and characterization of an OAS-TL gene from wild soybean. Our results are informative to further elucidate the function and evolution of OAS-TL in soybean.     

Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana

Chlorophyll (Chl) biosynthesis and degradation are the only biochemical processes on Earth that can be directly observed from satellites or other planets. The bulk of the Chls is found in the light-harvesting antenna complexes of photosynthetic organisms. Surprisingly little is known about the biosynthesis of Chl b, which is the second most abundant Chl pigment after Chl a. We describe here the expression and properties of the chlorophyllide a oxygenase gene (CAO) from Arabidopsis thaliana, which is apparently the key enzyme in Chl b biosynthesis. The recombinant enzyme produced in Escherichia coli catalyses an unusual two-step oxygenase reaction that is the 'missing link' in the chlorophyll cycle of higher plants.     

Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

Key message

This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L.

Abstract

Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a β-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

     

Molecular cloning and expression analyses of mitochondrial and plastidic isoforms of cysteine synthase (O-acetylserine(thiol)lyase) fromArabidopsis thaliana

Summary Cysteme synthase, the key enzyme for fixation of inorganic sulfide, catalyses the formation of cysteine from O-acetylserine and inorganic sulfide. Here we report the cloning of cDNAs encoding cysteine synthase isoforms fromArabidopsis thaliana. The isolated cDNA clones encode for a mitochondrial and a plastidic isoform of cysteine synthase (O-acetylserine (thiol)-lyase, EC 4.2.99.8), designated cysteine synthase C (AtCS-C, CSase C) and B (AtCS-B; CSase B), respectively.AtCS-C andAtCS-B, having lengths of 1569-bp and 1421-bp, respectively, encode polypeptides of 430 amino acids (45.8 kD) and of 392 amino acids ( 41.8 kD), respectively. The deduced amino acid sequences of the mitochondrial and plastidic isoforms exhibit high homology even with respect to the presequences. The predicted presequence of AtCS-C has a N-terminal extension of 33 amino acids when compared to the plastidic isoform. Northern blot analysis showed thatAtCS-C is higher expressed in roots than in leaves whereas the expression ofAtCS-B is stronger in leaves. Furthermore, gene expression of both genes was enhanced by sulfur limitation which in turn led to an increase in enzyme activity in crude extracts of plants. Expression of theAtCS-B gene is regulated by light. The mitochondrial, plastidic and cytosolic (Hesse and Altmann, 1995) isoforms of cysteine synthase ofArabidopsis are able to complement a cysteine synthasedeficient mutant ofEscherichia coli unable to grow on minimal medium without cysteine, indicating synthesis of functional plant proteins in the bacterium. Two lines of evidence proved thatAtCS-C encodes a mitochondrial form of cysteine synthase; first, import ofin vitro translation products derived from AtCS-C in isolated intact mitochondria and second, Western blot analysis of mitochondria isolated from transgenic tobacco plants expressing AtCS-C cDNA/c-myc DNA fusion protein.Abbreviations CSase cysteine synthase The nucleotide sequence data reported will appear in the EMBL Database under the accession numbers X81973 forAtCS-C and X81698 forAtCS-B.     

Identification of a gene family (kat) encoding kinesin-like proteins in Arabidopsis thaliana and the characterization of secondary structure of KatA

A gene family, designated kat, has been characterized in Arabidopsis thaliana by genomic Southern hybridization and nucleotide sequencing analysis. The kat gene family includes at least five members, named katA, katB, katC, katD and katE, whose products share appreciable sequence similarities in their presumptive ATP-binding and microtubule-binding motifs with known kinesin-like proteins. The carboxyl-terminal region of the KatA protein deduced from the nucleotide sequence of the cDNA clone has considerable homology with the mechanochemical motor domain of the kinesin heavy chain. The predicted secondary structure of the KatA protein indicates two globular domains separated by a long a helical coiled coil with heptad repeat structures, such as are commonly found in kinesin-like proteins.     

Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

Characterization of a ubiquitous expressed gene family encoding polygalacturonase in Arabidopsis thaliana

Pectin, as one of the major components of plant cell wall, has been implicated in many developmental processes occurring during plant growth. Among the different enzymes known to participate in the pectin structure modifications, polygalacturonase (PG) activity has been shown to be associated with fruit ripening, organ abscission and pollen grain development. Until now, sequence analyses of the deduced polypeptides of the plant PG genes allowed their grouping into three clades corresponding to genes involved in one of these three activities. In this study, we report the sequence of three genomic clones encoding PG in Arabidopsis thaliana. These genes, together with 16 other genes present in the databases form a large gene family, ubiquitously expressed, present on the five chromosomes with at least two gene clusters on chromosomes II and V, respectively. Phylogenetic analyses suggest that the A. thaliana gene family contains five classes of genes, with three of them corresponding to the previously defined clades. Comparison of positions and numbers of introns among the A. thaliana genes reveals structural conservation between genes belonging to the same class. The pattern of intron losses that could have given rise to the PG gene family is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a spliced mRNA. Following this event of intron loss, the acquisition of introns in novel positions is consistent with a mechanism of intron gain at proto-splice sites.