HCN Regulates Cellular Processes through Posttranslational Modification of Proteins by S-cyanylation.

Hydrogen cyanide (HCN) is coproduced with ethylene in plant cells and is primarily enzymatically detoxified by the mitochondrial ß-CYANOALANINE SYNTHASE (CAS-C1). Permanent or transient depletion of CAS-C1 activity in Arabidopsis (Arabidopsis thaliana) results in physiological alterations in the plant that suggest that HCN acts as a gasotransmitter molecule. Label-free quantitative proteomic analysis of mitochondrially enriched samples isolated from the wild type and cas-c1 mutant revealed significant changes in protein content, identifying 451 proteins that are absent or less abundant in cas-c1 and 353 proteins that are only present or more abundant in cas-c1 Gene ontology classification of these proteins identified proteomic changes that explain the root hairless phenotype and the altered immune response observed in the cas-c1 mutant. The mechanism of action of cyanide as a signaling molecule was addressed using two proteomic approaches aimed at identifying the S-cyanylation of Cys as a posttranslational modification of proteins. Both the 2-imino-thiazolidine chemical method and the direct untargeted analysis of proteins using liquid chromatography-tandem mass spectrometry identified a set of 163 proteins susceptible to S-cyanylation that included SEDOHEPTULOSE 1,7-BISPHOSPHATASE (SBPase), the PEPTIDYL-PROLYL CIS-TRANS ISOMERASE 20-3 (CYP20-3), and ENOLASE2 (ENO2). In vitro analysis of these enzymes showed that S-cyanylation of SBPase Cys74, CYP20-3 Cys259, and ENO2 Cys346 residues affected their enzymatic activity. Gene Ontology classification and protein-protein interaction cluster analysis showed that S-cyanylation is involved in the regulation of primary metabolic pathways, such as glycolysis, and the Calvin and S-adenosyl-Met cycles.

O-acetylserine(thio)lyase (OAS-TL) molecular expression in Pancratium maritimum L. (Amaryllidaceae) under salt stress.

MAIN CONCLUSION: Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein.

O-acetylserine sulfhydrylase A (CysK) is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This "cysteine synthase" complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional "moonlighting" activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Multiparameter behavioral analyses provide insights to mechanisms of cyanide resistance in Caenorhabditis elegans.

Environmental toxicants influence development, behavior, and ultimately survival. The nematode Caenorhabditis elegans has proven to be an exceptionally powerful model for toxicological studies. Here, we develop novel technologies to describe the effects of cyanide toxicity with high spatiotemporal resolution. Importantly, we use these methods to examine the genetic underpinnings of cyanide resistance. Caenorhabditis elegans that lack the EGL-9 oxygen sensing enzyme have been shown to be resistant to hydrogen cyanide (HCN) gas produced by the pathogen Pseudomonas aeruginosa PAO1. We demonstrate that the cyanide resistance exhibited by egl-9 mutants is completely dependent on the HIF-1 hypoxia-inducible factor and is mediated by the cysl-2 cysteine synthase, which likely functions in metabolic pathways that inactivate cyanide. Further, the expression of cysl-2 correlates with the degree of cyanide resistance exhibited in each genetic background. We find that each mutant exhibits similar relative resistance to HCN gas on plates or to aqueous potassium cyanide in microfluidic chambers. The design of the microfluidic devices, in combination with real-time imaging, addresses a series of challenges presented by mutant phenotypes and by the chemical nature of the toxicant. The microfluidic assay produces a set of behavioral parameters with increased resolution that describe cyanide toxicity and resistance in C. elegans, and this is particularly useful in analyzing subtle phenotypes. These multiparameter analyses of C. elegans behavior hold great potential as a means to monitor the effects of toxicants or chemical interventions in real time and to study the biological networks that underpin toxicant resistance.

Identification of aluminum-responsive proteins in rice roots by a proteomic approach: cysteine synthase as a key player in Al response.

Aluminum (Al) toxicity is a serious limitation to worldwide crop production. Rice is one of the most Al-tolerant crops and also serves as an important monocot model plant. This study aims to identify Al-responsive proteins in rice, based on evidence that Al resistance is an inducible process. Two Al treatment systems were applied in the study: Al3+-containing simple Ca solution culture and Al3+-containing complete nutrient solution culture. Proteins prepared from rice roots were separated by 2-DE. The 2-DE patterns were compared and the differentially expressed proteins were identified by MS. A total of 17 Al-responsive proteins were identified, with 12 of those being up-regulated and 5 down-regulated. Among the up-regulated proteins are copper/zinc superoxide dismutase (Cu-Zn SOD), GST, and S-adenosylmethionine synthetase 2, which are the consistently known Al-induced enzymes previously detected at the transcriptional level in other plants. More importantly, a number of other identified proteins including cysteine synthase (CS), 1-aminocyclopropane-1-carboxylate oxidase, G protein beta subunit-like protein, abscisic acid- and stress-induced protein, putative Avr9/Cf-9 rapidly elicited protein 141, and a 33 kDa secretory protein are novel Al-induced proteins. Most of these proteins are functionally associated with signaling transduction, antioxidation, and detoxification. CS, as consistently detected in both Al stress systems, was further validated by Western blot and CS activity assays. Moreover, the metabolic products of CS catalysis, i.e. both the total glutathione pool and reduced glutathione, were also significantly increased in response to Al stress. Taken together, our results suggest that antioxidation and detoxification ultimately related to sulfur metabolism, particularly to CS, may play a functional role in Al adaptation for rice.

Functional analysis of the cysteine synthase protein complex from plants: structural, biochemical and regulatory properties.

Cysteine synthesis in plants represents the final step of assimilatory sulfate reduction and the almost exclusive entry reaction of reduced sulfur into metabolism not only of plants, but also the human food chain in general. It is accomplished by the sequential reaction of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they form the hetero-oligomeric cysteine synthase complex (CSC). Recent evidence is reviewed that identifies the dual function of the CSC as a sensor and as part of a regulatory circuit that controls cellular sulfur homeostasis. Computational modeling of three-dimensional structures of plant SAT and OAS-TL based on the crystal structure of the corresponding bacterial enzymes supports quaternary conformations of SAT as a dimer of trimers and OAS-TL as a homodimer. These findings suggest an overall alpha6beta4 structure of the subunits of the plant CSC. Kinetic measurements of CSC dissociation triggered by the reaction intermediate O-acetylserine as well as CSC stabilization by sulfide indicate quantitative reactions that are suited to fine-tune the equilibrium between free and associated CSC subunits. In addition, in vitro data show that SAT requires binding to OAS-TL for full activity, while at the same time bound OAS-TL becomes inactivated. Since OAS concentrations inside cells increase upon sulfate deficiency, whereas sulfide concentrations most likely decrease, these data suggest the dissociation of the CSC in vivo, accompanied by inactivation of SAT and activation of OAS-TL function in their free homo-oligomer states. Biochemical evidence describes this protein-interaction based mechanism as reversible, thus closing the regulatory circuit. The properties of the CSC and its subunits are therefore consistent with models of positive regulation of sulfate uptake and reduction in plants by OAS as well as a demand-driven repression/de-repression by a sulfur intermediate, such as sulfide.

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum.

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.

Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure, function, and stability.

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.

Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana.

Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (K(D) = 25 +/- 4 x 10(-9) m), based on a reliable A + B <--> AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 +/- 4 microm O-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded a K(m) value of 58 +/- 7 microm O-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function.

Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants.

Cysteine (Cys) synthase [O-acetyl-L-Ser(thiol)-lyase, EC 4.2.99.8; CSase] is responsible for the final step in biosynthesis of Cys. Transgenic tobacco (Nicotiana tabacum; F(1)) plants with enhanced CSase activities in the cytosol and in the chloroplasts were generated by cross-fertilization of two transformants expressing cytosolic CSase or chloroplastic CSase. The F(1) transgenic plants were highly tolerant to toxic sulfur dioxide and sulfite. Upon fumigation with 0.1 microL L(-1) sulfur dioxide, the Cys and glutathione contents in leaves of F(1) plants were increased significantly, but not in leaves of non-transformed control plants. Furthermore, the leaves of F(1) plants exhibited the increased resistance to paraquat, a herbicide generating active oxygen species.

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.