Pyridoxal Phosphate-dependent Enzymes Research Articles

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and β-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Edodin: A New Type of Toxin from Shiitake Mushroom (Lentinula edodes) That Inactivates Mammalian Ribosomes.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.

Structure and function of the pyridoxal 5′-phosphate-dependent (PLP) threonine deaminase IlvA1 from Pseudomonas aeruginosa PAO1

IlvA1, a pyridoxal phosphate-dependent (PLP) enzyme, catalyzes the deamination of l-threonine and l-serine to yield 2-ketobutyric acid or pyruvate. To gain insights into the function of IlvA1, we determined its crystal structure from Pseudomonas aeruginosa to 2.3 Å. Density for a 2-ketobutyric acid product was identified in the active site and a putative allosteric site. Activity and substrate binding assays confirmed that IlvA1 utilizes l-threonine, l-serine, and L-allo-threonine as substrates. The enzymatic activity is regulated by the end products l-isoleucine and l-valine. Additionally, the efficiency of d-cycloserine and l-cycloserine inhibitors on IlvA1 enzymatic activity was examined. Notably, site-directed mutagenesis confirmed the active site residues and revealed that Gln165 enhances the enzyme activity, emphasizing its role in substrate access. This work provides crucial insights into the structure and mechanism of IlvA1 and serves as a starting point for further functional and mechanistic studies of the threonine deaminase in P. aeruginosa.

Structure of the Oxygen, Pyridoxal Phosphate-Dependent Capuramycin Biosynthetic Protein Cap15.

Pyridoxal phosphate-dependent enzymes able to use oxygen as a co-substrate have emerged in multiple protein families. Here, we use crystallography to solve the 2.40 Å resolution crystal structure of Cap15, a nucleoside biosynthetic enzyme that catalyzes the oxidative decarboxylation of glycyl uridine. Our structural study captures the internal aldimine, pinpointing the active site lysine as K230 and showing the site of phosphate binding. Our docking studies reveal how Cap15 is able to catalyze a stereoselective deprotonation reaction, and bioinformatic analysis reveals active site residues that distinguish Cap15 from the structurally related d-glucosaminate-6-phosphate ammonia lyase and l-seryl-tRNA(Sec) selenium transferase (SelA). Our work provides the structural basis for further mechanistic investigation of a unique biosynthetic enzyme and provides a blueprint for understanding how oxygen reactivity emerged in the SelA-like protein family.

Proteomic and Phenotypic Analyses of a Putative YggS Family Pyridoxal Phosphate-Dependent Enzyme in Acidovorax citrulli.

Acidovorax citrulli (Ac) is a phytopathogenic bacterium that causes bacterial fruit blotch (BFB) in cucurbit crops, including watermelon. However, there are no effective methods to control this disease. YggS family pyridoxal phosphate-dependent enzyme acts as a coenzyme in all transamination reactions, but its function in Ac is poorly understood. Therefore, this study uses proteomic and phenotypic analyses to characterize the functions. The Ac strain lacking the YggS family pyridoxal phosphate-dependent enzyme, AcΔyppAc(EV), virulence was wholly eradicated in geminated seed inoculation and leaf infiltration. AcΔyppAc(EV) propagation was inhibited when exposed to L-homoserine but not pyridoxine. Wild-type and mutant growth were comparable in the liquid media but not in the solid media in the minimal condition. The comparative proteomic analysis revealed that YppAc is primarily involved in cell motility and wall/membrane/envelop biogenesis. In addition, AcΔyppAc(EV) reduced biofilm formation and twitching halo production, indicating that YppAc is involved in various cellular mechanisms and possesses pleiotropic effects. Therefore, this identified protein is a potential target for developing an efficient anti-virulence reagent to control BFB.

Targeting the liver to treat the eye.

Over the last two decades, gene therapy has given hope of potential cure for many rare diseases. In the simplest form, gene therapy is the transfer or editing of a genetic material to cure a disease via nonviral or viral vehicles. Gene therapy can be performed either in vivo by injecting a vector carrying the gene or tools for gene editing directly into a tissue or into the systemic circulation, or ex vivo when patient cells are genetically modified outside of the body and then introduced back into the patient (Yilmaz et al, 2022). Adeno-associated viral vectors (AAV) have been the vectors of choice for in vivo gene therapy. There has been a lot of promising research on the development of novel tissue and cell-specific serotypes in order to improve efficacy and safety for clinical applications (Kuzmin et al, 2021). In this issue of EMBO Molecular Medicine, Boffa and colleagues present a novel AAV-based liver-directed gene therapy for ornithine aminotransferase deficiency.

Knockdown of SHMT2 enhances the sensitivity of gastric cancer cells to radiotherapy through the Wnt/β-catenin pathway.

Gastric cancer (GC) is one of the most common malignant tumors. The mechanism of GC radioresistance and new radiosensitizers must be revealed and developed to treat GC. Serine hydroxymethyltransferase 2 (SHMT2) is responsible for encoding the mitochondrial form of the pyridoxal phosphate-dependent enzyme. SHMT2 plays a critical role in several types of cancers, while its possible effect on the radiological resistance in GC is still unclear. In this study, we investigated the role of SHMT2 in the radiological resistance of GC. Our data confirmed that SHMT2 was highly expressed in radiation-resistant GC cells. SHMT2 reduced the radiosensitivity of GC cells. In addition, SHMT2 is involved in radiation-induced GC cell apoptosis. Further, SHMT2 regulated the Wnt/β-catenin pathway, therefore reducing the radiosensitivity of GC cells in vivo. In conclusion, we revealed that depletion of SHMT2 enhanced the sensitivity of GC cells to interventional radiotherapy through the Wnt/β-catenin pathway.

Tailored Pyridoxal Probes Unravel Novel Cofactor-Dependent Targets and Antibiotic Hits in Critical Bacterial Pathogens.

Unprecedented bacterial targets are urgently needed to overcome the resistance crisis. Herein we systematically mine pyridoxal phosphate‐dependent enzymes (PLP‐DEs) in bacteria to focus on a target class which is involved in crucial metabolic processes. For this, we tailored eight pyridoxal (PL) probes bearing modifications at various positions. Overall, the probes exceeded the performance of a previous generation and provided a detailed map of PLP‐DEs in clinically relevant pathogens including challenging Gram‐negative strains. Putative PLP‐DEs with unknown function were exemplarily characterized via in‐depth enzymatic assays. Finally, we screened a panel of PLP binders for antibiotic activity and unravelled the targets of hit molecules. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP‐dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP‐DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.

YggS Encoding Pyridoxal 5'-Phosphate Binding Protein Is Required for Acidovorax citrulli Virulence.

Bacterial fruit blotch, caused by seed-borne pathogen Acidovorax citrulli, poses a serious threat to the production of cucurbits globally. Although the disease can cause substantial economic losses, limited information is available about the molecular mechanisms of virulence. This study identified that, a random transposon insertion mutant impaired in the ability to elicit a hypersensitive response on tobacco. The disrupted gene in this mutant was determined to be Aave_0638, which is predicted to encode a YggS family pyridoxal phosphate-dependent enzyme. YggS is a highly conserved protein among multiple organisms, and is responsible for maintaining the homeostasis of pyridoxal 5′-phosphate and amino acids in cells. yggS deletion mutant of A. citrulli strain XjL12 displayed attenuated virulence, delayed hypersensitive response, less tolerance to H2O2 and pyridoxine, increased sensitivity to antibiotic β-chloro-D-alanine, and reduced swimming. In addition, RNA-Seq analysis demonstrated that yggS was involved in regulating the expression of certain pathogenicity-associated genes related to secretion, motility, quorum sensing and oxidative stress response. Importantly, YggS significantly affected type III secretion system and its effectors in vitro. Collectively, our results suggest that YggS is indispensable for A.citrulli virulence and expands the role of YggS in the biological processes.

The role of the 'sphingoid motif' in shaping the molecular interactions of sphingolipids in biomembranes.

Sphingolipids can be differentiated from other membrane lipids by the distinctive chemistry of the sphingoid long chain base (LCB), which is generated by the condensation of an amino acid (normally but not always serine) and a fatty acyl CoA (normally palmitoyl CoA) by the pyridoxal phosphate-dependent enzyme, serine palmitoyl transferase (SPT). The first five carbon atoms of the sphingoid LCB, herein defined as the 'sphingoid motif', are largely responsible for the unique chemical and biophysical properties of sphingolipids since they can undergo a relatively large number (compared to other lipid species) of molecular interactions with other membrane lipids, via hydrogen-bonding, charge-pairing, hydrophobic and van der Waals interactions. These interactions are responsible, for instance, for the association of sphingolipids with cholesterol in the membrane lipid bilayer. Here, we discuss some of the unique properties of this sphingoid motif, and in addition to outlining how this structural motif drives intra-bilayer interactions, discuss the atomic details of the interactions with two critical players in the biosynthetic pathway, namely SPT, and the ceramide transport protein, CERT. In the former, the selectivity of sphingolipid synthesis relies on a hydrogen bond interaction between Lys379 of SPTLC2 and the l-serine sidechain hydroxyl moiety. In the latter, the entire sphingoid motif is stereoselectively recognized by a hydrogen-bonding network involving all three sphingoid motif heteroatoms. The remarkable selectivity of these interactions, and the subtle means by which these interactions are modified and regulated in eukaryotic cells raises a number of challenging questions about the generation of these proteins, and of their interactions with the sphingoid motif in evolutionary history.

Nutritional Factors That Affect the Formation of 5-Aminolevulinic Acid, a Key Intermediate of Heme Biosynthesis.

5-Aminolevulinic acid (ALA) is a key intermediate of heme biosynthesis, which is an essential component of the respiratory chain. Therefore, nutrients that affect ALA biosynthesis eventually affect ATP production, which is the basis of mitochondrial function. Although the effects of various non-nutrient components that affect ALA after biosynthesis have been reported, there are few reports on the effects of dietary amino acids/protein on ALA formation and the effects of dietary vitamins that are involved in amino acid metabolism. In mitochondria, ALA is synthesized from succinyl-CoA and glycine by the pyridoxal phosphate-dependent enzyme ALA synthase [EC 2.3.1.37]. In this study, the effects of dietary amino acids/protein and vitamins on the amount of ALA synthesized were investigated using mice, rats, and cultured cells. Amounts of ALA in plasma and urine, and porphyrins in plasma increased with increasing protein intake. Vitamin B1 insufficiency did not affect ALA synthesis. Vitamin B6 insufficiency increased the amount of ALA synthesized, while niacin deficiency markedly reduced ALA synthesis. Thus, for heme synthesis, an essential biological substance for life, the amounts of amino acids, as well as the pathways metabolizing amino acids to glycine and succinyl-CoA are very important. Specifically, it is important that niacin is associated with the formation of glycine and succinyl-CoA from amino acids.

Diversity of the Tryptophanase Gene and Its Evolutionary Implications in Living Organisms.

Tryptophanase encoded by the gene tnaA is a pyridoxal phosphate-dependent enzyme that catalyses the conversion of tryptophan to indole, which is commonly used as an intra- and interspecies signalling molecule, particularly by microbes. However, the production of indole is rare in eukaryotic organisms. A nucleotide and protein database search revealed tnaA is commonly reported in various Gram-negative bacteria, but that only a few Gram-positive bacteria and archaea possess the gene. The presence of tnaA in eukaryotes, particularly protozoans and marine organisms, demonstrates the importance of this gene in the animal kingdom. Here, we document the distribution of tnaA and its acquisition and expansion among different taxonomic groups, many of which are usually categorized as non-indole producers. This study provides an opportunity to understand the intriguing role played by tnaA, and its distribution among various types of organisms.

KYNU, a novel potential target that underpins CD44-promoted breast tumour cell invasion.

Using a validated tetracycline‐off‐inducible CD44 expression system in mouse model, we have previously demonstrated that the hyaluronan (HA) receptor CD44 promotes breast cancer (BC) metastasis to the liver. To unravel the mechanisms that underpin CD44‐promoted BC cell invasion, RNA samples were isolated from two cell models: (a) a tetracycline (Tet)‐Off‐regulated expression system of the CD44s in MCF‐7 cells and; (b) as a complementary approach, the highly metastatic BC cells, MDA‐MB‐231, were cultured in the presence and absence of 50 µg/mL of HA. Kynureninase (KYNU), identified by Microarray analysis, was up‐regulated by 3‐fold upon induction and activation of CD44 by HA; this finding suggests that KYNU is a potential novel transcriptional target of CD44‐downtstream signalling. KYNU is a pyridoxal phosphate (PLP) dependent enzyme involved in the biosynthesis of NAD cofactors from tryptophan that has been associated with the onset and development of BC. This review will attempt to identify and discuss the findings supporting this hypothesis and the mechanisms linking KYNU cell invasion via CD44.

Quorum Sensing System-Regulated Proteins Affect the Spoilage Potential of Co-cultured Acinetobacter johnsonii and Pseudomonas fluorescens From Spoiled Bigeye Tuna (Thunnus obesus) as Determined by Proteomic Analysis.

Food spoilage by certain species of bacteria is reported to be regulated by quorum sensing (QS). Acinetobacter johnsonii and Pseudomonas fluorescens, the major specific spoilage organisms, are found to be limited in their QS and co-culture interactions. The aim of this study was to determine how QS-regulated proteins affect the spoilage potential of co-cultured A. johnsonii and P. fluorescens obtained from spoiled bigeye tuna (Thunnus obesus) using a proteomics approach. The A. johnsonii, P. fluorescens, and their co-culture tested the N-acyl-homoserine lactone (AHL) activities using reporter Chromobacterium violaceum CV026 and LC-MS/MS in qualitative and quantitative approaches, respectively. These latter showed that, of the 470 proteins and 444 proteins in A. johnsonii (A) and P. fluorescens (P), respectively, 80 were significantly up-regulated and 97 were significantly down-regulated in A vs. AP, whereas 90 were up-regulated and 65 were down-regulated in P vs. AP. The differentially expressed proteins included the AI-2E family transporter OS, 50S ribosomal protein L3, thioredoxin reductase OS, cysteine synthase CysM OS, DNA-binding response regulator, and amino acid ABC transporter ATPase OS. The cellular process (GO:0009987), metabolic process (GO:0008152), and single-organism process (GO:0044699) were classified into the gene ontology (GO) term. In addition, energy production and conversion, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, post-translational modification, protein turnover, and chaperones were distributed into the clusters of orthologous groups of proteins (COG) terms. The KEGG pathways revealed that 84 and 77 differentially expressed proteins were divided into 20 KEGG pathways in A vs. AP and P vs. AP, respectively, and amino acid metabolism, carbohydrate metabolism, energy metabolism, and translation were significantly enriched. Proteins that correlated with the spoilage-related metabolic pathways, including thioredoxin reductase OS, cysteine synthase OS, and pyridoxal phosphate-dependent enzyme family protein OS, were identified. AI-2E family transporter OS and LuxR family transcriptional regulator OS were identified that related to the QS system. These findings provide a differential proteomic profile of co-culture in A. johnsonii and P. fluorescens, and have potential applications in QS and the regulation of spoilage potential.

An unusual metal-bound 4-fluorothreonine transaldolase from Streptomyces sp. MA37 catalyses promiscuous transaldol reactions

β-Hydroxy-α-amino acids (βH-AAs) are key components of many bioactive molecules as well as exist as specialised metabolites. Among these βH-AAs, 4-fluorothreonine (4-FT) is the only naturally occurring fluorinated AA discovered thus far. Here we report overexpression and biochemical characterisation of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA), a homologue of FTase previously identified in the biosynthesis of 4-FT in S. cattleya. FTaseMA displays considerable substrate plasticity to generate 4-FT as well as other β-hydroxy-α-amino acids with various functionalities at C4 position, giving the prospect of new chemo-enzymatic applications. The enzyme has a hybrid of two catalytic domains, serine hydroxymethyltransferase (S) and aldolase (A). Site-directed mutagenesis allowed the identification of the key residues of FTases, suggesting that the active site of A domain has a historical reminiscent feature in metal-dependent aldolases. Elemental analysis demonstrated that FTaseMA is indeed a Zn2+-dependent enzyme, the first example of pyridoxal phosphate (PLP) enzyme family fused with a metal-binding domain carrying out a distinct catalytic role. Finally, FTaseMA showed divergent evolutionary origin with other PLP dependent enzymes.

Off to a slow start: Analyzing lag phases and accelerating rates in steady-state enzyme kinetics

Steady-state enzyme kinetics typically relies on the measurement of ‘initial rates’, obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or ‘lag phase’. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s−1.

Pyridoxal 5'-phosphate dependent reactions: Analyzing the mechanism of aspartate aminotransferase.

Enzyme catalysis is the primary activity in energy and information metabolism and enzyme cofactors are key to the catalytic ability of most enzymes. Pyridoxal 5'-phosphate (PLP) cofactor, derived from Vitamin B6, is widely distributed in nature and has significant latitude in catalytic diversity. X-ray crystallography has revealed the structures of diverse PLP dependent enzymes from multiple families. But these structures are incomplete, lacking the positions of protons essential for understanding enzymatic mechanisms. Here, we review the diversity of PLP and discuss the use of neutron crystallography and joint X-ray/neutron refinement of Fold Type I aspartate aminotransferase to visualize the positions of protons in both the internal and external aldimine forms. Strategies used to prepare extremely large crystals required for neutron diffraction and the approach to data refinement including the PLP cofactor are discussed. The observed positions of protons, including one located in a previously unknown low-barrier hydrogen bond, have been used to create more accurate models for computational analysis. The results revealed a new mechanism for the transaminase reaction where hyperconjugation is key to reducing the energy barrier which finally provides a clear explanation of the Dunathan alignment.

Hydrogen sulfide formation in experimental model of acute pancreatitis.

Acute pancreatitis (AP) is a disease defined as acute or chronic inflammatory process of the pancreas characterized by premature activation of digestive enzymes within the pancreatic acinar cells and causing pancreatic auto-digestion. In mammalian tissues, H2S is synthesized endogenously from L-cysteine in regulated enzymatic pathways catalyzed by pyridoxal phosphate-dependent enzymes: cystathionine beta - synthase (CBS), gamma - cystathionase (CTH) and cysteine aminotransferase (CAT) coupled with 3-mercaptopyruvate sulfurtransferase (MPST). In the mitochondria, hydrogen sulfide is oxidized to sulfite, which is then converted to thiosulfate (sulfane sulfur-containing compound) by thiosulfate sulfurtransferase (rhodanese; TST). The activity and the expression of CBS, CTH, MPST, and TST have been determined in vivo in pancreas of control rats, rats with acute pancreatitis and sham group. Levels of low-molecular sulfur compounds such as reduced and oxidized glutathione, cysteine, cystine and cystathionine were also determined. The study showed the significant role of MPST in H2S metabolism in pancreas. Stress caused by the surgery (sham group) and AP cause a decrease in H2S production associated with a decrease of MPST activity and expression. Markedly higher level of cysteine in the AP pancreas may be caused by a reduced rate of cysteine consumption in reaction catalyzed by MPST but it can also be a sign of the processes of proteolysis occurring in the changed tissue.

Metagenomic analysis of relationships between the denitrification process and carbon metabolism in a bioaugmented full-scale tannery wastewater treatment plant.

The goal of this study was to investigate the relationship between the denitrification process and carbon metabolism in a full-scale tannery wastewater treatment plant bioaugmented with the microbial consortium BM-S-1. The metagenomic analysis of the microbial community showed that Brachymonas denitrificans, a known denitrifier, was present at a high level in the treatment stages of buffering (B), primary aeration (PA), and sludge digestion (SD). The occurrences of the amino acid-degrading enzymes alpha ketoglutarate dehydrogenase (α-KGDH) and tryptophan synthase were highly correlated with the presence of denitrification genes, such as napA, narG, nosZ and norB. The occurrence of glutamate dehydrogenase (GDH) was also highly paralleled with the occurrence of denitrification genes such as napA, narG, and norZ. The denitrification genes (nosZ, narG, napA, norB and nrfA) and amino acid degradation enzymes (tryptophan synthase, α-KGDH and pyridoxal phosphate dependent enzymes) were observed at high levels in B. This indicates that degradation of amino acids and denitrification of nitrate may potentially occur in B. The high concentrations of the fatty acid degradation enzyme groups (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and β-ketothiolase) were observed together with the denitrification genes, such as napA, narG and nosZ. Phospholipase/carboxylesterase, enoyl-CoA hydratase/isomerase, acyl-CoA dehydrogenase, phenylacetate degradation enzyme and 3-hydroxyacyl-CoA dehydrogenase 2 were also dominant in B. All these results clearly indicate that the denitrification pathways are potentially linked to the degradation pathways of amino acids and fatty acids whose degradation products go through the TCA cycle, generating the NADH that is used as electron donors for denitrification.

Hydrogen sulfide may function downstream of hydrogen peroxide in CdCl2-induced stomatal closure in Vigna radiata L.

Pharmacological, spectrophotographic and laser scanning confocal microscopy (LSCM) approaches were used to investigate the roles of hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) played in signaling transduction of stomatal closure induced by CdCl2 in Vigna radiata L. In the present study, CdCl2 could induce stomatal closure in a dose- and time-dependent manner, which was inhibited by H2S modulators hypotaurine (HT), aminooxy acetic acid (AOA), hydroxylamine (NH2OH), potassium pyruvate (C3H3KO3), ammonia (NH3), H2O2 modulators ascorbic acid (ASA), catalase (CAT), diphenylene iodonium (DPI) and salicylhydroxamic acid (SHAM). In addition, both the content of H2S, activity of L-/D-cysteine desulfhydrase (L-/D-CDes and pyridoxalphosphate-dependent enzyme) in leaves of V. radiata, and the level of H2O2 in guard cells significantly increased under CdCl2 stress, which were inhibited by H2S and H2O2 modulators, respectively. H2O2 modulators could suppress CdCl2-induced increase of H2S levels, L-/D-CDes activity and stomatal closure in the leaves of V. radiata. However, H2S modulators had no effect on the accumulation of CdCl2-induced H2O2 in guard cells of V. radiata. From these data, it can be deduced that both H2S and H2O2 can mediate CdCl2-induced stomatal closure in V. radiata L., and H2S acts downstream than H2O2 in such process.