Characterization of glutathione-specific gamma glutamyl cyclotransferase (ChaC) in Bombyx mori.

Glutathione (GSH) contributes to redox maintenance and detoxification of various xenobiotic and endogenous substances. γ-glutamyl cyclotransferase (ChaC) is involved in GSH degradation. However, the molecular mechanism underlying GSH degradation in silkworms (Bombyx mori) remains unknown. Silkworms are lepidopteran insects that are considered to be an agricultural pest model. We aimed to examine the metabolic mechanism underlying GSH degradation mediated by B. mori ChaC and successfully identified a novel ChaC gene in silkworms (herein, bmChaC). The amino acid sequence and phylogenetic tree revealed that bmChaC was closely related to mammalian ChaC2. We overexpressed recombinant bmChaC in Escherichia coli, and the purified bmChaC showed specific activity toward GSH. Additionally, we examined the degradation of GSH to 5-oxoproline and cysteinyl glycine via liquid chromatography-tandem mass spectrometry. Quantitative real-time polymerase chain reaction revealed that bmChaC mRNA expression was observed in various tissues. Our results suggest that bmChaC participates in tissue protection via GSH homeostasis. This study provides new insights into the activities of ChaC and the underlying molecular mechanisms that can aid the development of insecticides to control agricultural pests.

An omega-class glutathione S-transferase in the brown planthopper Nilaparvata lugens exhibits glutathione transferase and dehydroascorbate reductase activities.

A complementary DNA that encodes an omega-class glutathione S-transferase (GST) of the brown planthopper, Nilaparvata lugens (nlGSTO), was isolated by reverse transcriptase polymerase chain reaction. A recombinant protein (nlGSTO) was obtained via overexpression in the Escherichia coli cells and purified. nlGSTO catalyzes the biotransformation of glutathione with 1-chloro-2,4-dinitrobenzene, a general substrate for GST, as well as with dehydroascorbate to synthesize ascorbate. Mutation experiments revealed that putative substrate-binding sites, including Phe28, Cys29, Phe30, Arg176, and Lue225, were important for glutathione transferase and dehydroascorbate reductase activities. As ascorbate is a reducing agent, nlGSTO may participate in antioxidant resistance.

Catechol O-methyltransferase homologs in Schizosaccharomyces pombe are response factors to alkaline and salt stress.

How cells of the fission yeast Schizosaccharomyces pombe respond to alkaline stress is not well understood. Here, to elucidate the molecular mechanism underlying the alkaline stress response in S. pombe, we performed DNA microarray analysis. We found that a homolog of human catechol O-methyltransferase 2 (COMT2) is highly upregulated in S. pombe cells exposed to alkaline conditions. We designated the S. pombe homolog as cmt2+ and also identified its paralog, cmt1+, in the S. pombe genome. Reverse transcription PCR confirmed that both cmt1+ and cmt2+ are upregulated within 1 h of exposure to alkaline stress and downregulated within 30 min of returning to an acidic environment. Moreover, we verified that recombinant Cmt proteins exhibit catechol O-methyltransferase activity. To further characterize the expression of cmt1+ and cmt2+, we carried out an EGFP reporter assay using their promoter sequences, which showed that both genes respond not only to alkaline but also to salt stress. Collectively, our findings indicate that the cmt promoter might be an advantageous expression system for use in S. pombe under alkaline culture conditions.

Functional Analysis of an Epsilon-Class Glutathione S-Transferase From Nilaparvata lugens (Hemiptera: Delphacidae).

Glutathione conjugation is a crucial step in xenobiotic detoxification. In the current study, we have functionally characterized an epsilon-class glutathione S-transferase (GST) from a brown planthopper Nilaparvata lugens (nlGSTE). The amino acid sequence of nlGSTE revealed approximately 36-44% identity with epsilon-class GSTs of other species. The recombinant nlGSTE was prepared in soluble form by bacterial expression and was purified to homogeneity. Mutation experiments revealed that the putative substrate-binding sites, including Phe107, Arg112, Phe118, and Phe119, were important for glutathione transferase activity. Furthermore, inhibition study displayed that nlGSTE activity was affected by insecticides, proposing that, in brown planthopper, nlGSTE could recognize insecticides as substrates.

Nitrated cyclic GMP modulates guard cell signaling in Arabidopsis.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-5'-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells.

Structural characterization of the catalytic site of a Nilaparvata lugens delta-class glutathione transferase.

Glutathione transferases (GSTs) are a major class of detoxification enzymes that play a central role in the defense against environmental toxicants and oxidative stress. Here, we studied the crystal structure of a delta-class glutathione transferase from Nilaparvata lugens, nlGSTD, to gain insights into its catalytic mechanism. The structure of nlGSTD in complex with glutathione, determined at a resolution of 1.7Å, revealed that it exists as a dimer and its secondary and tertiary structures are similar to those of other delta-class GSTs. Analysis of a complex between nlGSTD and glutathione showed that the bound glutathione was localized to the glutathione-binding site. Site-directed mutagenesis of nlGSTD mutants indicated that amino acid residues Ser11, His52, Glu66, and Phe119 contribute to catalytic activity.

Novel silkworm (Bombyx mori) sulfotransferase swSULT ST3 is involved in metabolism of polyphenols from mulberry leaves.

Polyphenols in plants are important for defense responses against microorganisms, insect herbivory, and control of feeding. Owing to their antioxidant, anti-cancer, and anti-inflammatory activities, their importance in human nutrition has been acknowledged. However, metabolism of polyphenols derived from mulberry leaves in silkworms (Bombyx mori) remains unclear. Sulfotransferases (SULT) are involved in the metabolism of xenobiotics and endogenous compounds. The purpose of this study is to investigate the metabolic mechanism of polyphenols mediated by B. mori SULT. Here, we identified a novel SULT in silkworms (herein, swSULT ST3). Recombinant swSULT ST3 overexpressed in Escherichia coli effectively sulfated polyphenols present in mulberry leaves. swSULT ST3 showed high specific activity toward genistein among the polyphenols. Genistein-7-sulfate was produced by the activity of swSULT ST3. Higher expression of swSULT ST3 mRNA was observed in the midgut and fat body than in the hemocytes, testis, ovary, and silk gland. Polyphenols inhibited the aldo-keto reductase detoxification of reactive aldehydes from mulberry leaves, and the most noticeable inhibition was observed with genistein. Our results suggest that swSULT ST3 plays a role in the detoxification of polyphenols, including genistein, and contributes to the effects of aldo-keto reductase in the midgut of silkworms. This study provides new insight into the functions of SULTs and the molecular mechanism responsible for host plant selection in lepidopteran insects.

Structural analysis of α1,3-linked galactose-containing oligosaccharides in Schizosaccharomyces pombe mutants harboring single and multiple α-galactosyltransferase genes disruptions.

In the fission yeast Schizosaccharomyces pombe, galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases in the lumen of the Golgi apparatus. In S. pombe, the major in vitro α1,2-galactosyltransferase activity has been purified, the gma12(+) gene has been cloned, and three α-galactosyltransferase genes (gmh1(+)-gmh3(+)) have also been partially characterized. In this study, we found three additional uncharacterized genes with homology to gmh1(+) (gmh4(+)-gmh6(+)) in the fission yeast genome sequence. All possible single disruption mutants and the septuple disruption strain were constructed and characterized. The electrophoretic mobility of acid phosphatase prepared from gma12Δ, gmh2Δ, gmh3Δ and gmh6Δ mutants was higher than that from wild type, indicating that Gma12p, Gmh2p, Gmh3p and Gmh6p are required for the galactosylation of N-linked oligosaccharides. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides from each single mutant showed that Gma12p, Gmh2p and Gmh6p are involved in galactosylation of O-linked oligosaccharides. The septuple mutant exhibited similar drug and temperature sensitivity as a gms1Δ mutant that is incapable of galactosylation. Oligosaccharide structural analysis based on HPLC and methylation analysis revealed that the septuple mutant still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating that an unknown α1,3-galactosyltransferase activity was still present in the septuple mutant.

Investigation of the active site of an unclassified glutathione transferase in Bombyx mori by alanine scanning.

Glutathione transferase (GST) is an important class of detoxification enzymes that are vital for defense against various xenobiotics and cellular oxidative stress. Previously, we had reported an unclassified glutathione transferase 2 in Bombyx mori (bmGSTu2) to be responsible for detoxifying diazinon. In this study, we aimed to identify the amino acid residues that constitute a hydrogen-bonding network important for GST activity. Site-directed mutagenesis of bmGSTu2 suggested that residues Asn102, Pro162, and Ser166 contribute to its catalytic activity.

Guard cell photosynthesis is crucial in abscisic acid-induced stomatal closure.

Reactive oxygen species (ROS) are ubiquitous signaling molecules involved in diverse physiological processes, including stomatal closure. Photosynthetic electron transport (PET) is the main source of ROS generation in plants, but whether it functions in guard cell signaling remains unclear. Here, we assessed whether PET functions in abscisic acid (ABA) signaling in guard cells. ABA-elicited ROS were localized to guard cell chloroplasts in Arabidopsis thaliana, Commelina benghalensis, and Vicia faba in the light and abolished by the PET inhibitors 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea and 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. These inhibitors reduced ABA-induced stomatal closure in all three species, as well as in the NADPH oxidase-lacking mutant atrboh D/F. However, an NADPH oxidase inhibitor did not fully eliminate ABA-induced ROS in the chloroplasts, and ABA-induced ROS were still observed in the guard cell chloroplasts of atrboh D/F. This study demonstrates that ROS generated through PET act as signaling molecules in ABA-induced stomatal closure and that this occurs in concert with ROS derived through NADPH oxidase.

Characterisation of a diazinon-metabolising glutathione S-transferase in the silkworm Bombyx mori by X-ray crystallography and genome editing analysis.

Previously, we found an unclassified glutathione S-transferase 2 (bmGSTu2) in the silkworm Bombyx mori that conjugates glutathione to 1-chloro-2,4-dinitrobenzene and also metabolises diazinon, an organophosphate insecticide. Here, we provide a structural and genome-editing characterisation of the diazinon-metabolising glutathione S-transferase in B. mori. The structure of bmGSTu2 was determined at 1.68 Å by X-ray crystallography. Mutation of putative amino acid residues in the substrate-binding site showed that Pro13, Tyr107, Ile118, Phe119, and Phe211 are crucial for enzymatic function. bmGSTu2 gene disruption resulted in a decrease in median lethal dose values to an organophosphate insecticide and a decrease in acetylcholine levels in silkworms. Taken together, these results indicate that bmGSTu2 could metabolise an organophosphate insecticide. Thus, this study provides insights into the physiological role of bmGSTu2 in silkworms, detoxification of organophosphate insecticides, and drug targets for the development of a novel insecticide.

Effects of water temperature and light intensity on the acute toxicity of herbicide thiobencarb to a green alga, Raphidocelis subcapitata.

The present study investigated how principal environmental factors such as temperature and light intensity change the toxicological properties of thiobencarb (TB) herbicide to the green alga, Raphidocelis subcapitata. At first, we investigated the inhibitory effect of TB (0, 15.6, 31.2, 62.4, and 125 µg L-1) on growth of R. subcapitata at five temperatures (10, 15, 20, 25, or 30 °C) for 144 h exposure and calculated 72- and 144-h effective concentration values (EC10, 20, and EC50) for growth rate. All EC values significantly decreased with an increasing temperature. The maximum quantum yield of photosystem II in R. subcapitata exposed to 125 µg L-1 of TB was also significantly inhibited with increased temperature. These physiological effects could explain the lower EC values at high temperatures. Then, single and interactive effects of TB, temperature, and light intensity on growth rate were investigated by three-way of analysis of variance. As a result, single and interactive effects were detected in all explanatory variables. These results suggest that temperature and light intensity change the acute toxicity parameter in R. subcapitata exposed to TB and must be considered in evaluating the risk of TB.

Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori.

The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription-polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides.

Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella.

cDNA encoding an unclassified glutathione S-transferase (GST) of the diamondback moth, Plutella xylostella, was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and the amino acid sequence deduced, revealing 67%-73% identities with unclassified GSTs from other organisms. A recombinant protein was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. The enzyme was capable to catalyze the transformation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid with glutathione. A competition assay revealed that GST activity was inhibited by insecticides, suggesting that the enzyme could contribute to insecticide metabolism in the diamondback moth.

Glutathione-binding site of a bombyx mori theta-class glutathione transferase.

The glutathione transferase (GST) superfamily plays key roles in the detoxification of various xenobiotics. Here, we report the isolation and characterization of a silkworm protein belonging to a previously reported theta-class GST family. The enzyme (bmGSTT) catalyzes the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)-propane, and 4-nitrophenethyl bromide. Mutagenesis of highly conserved residues in the catalytic site revealed that Glu66 and Ser67 are important for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTT and into the metabolism of exogenous chemical agents.

Elevated water temperature reduces the acute toxicity of the widely used herbicide diuron to a green alga, Pseudokirchneriella subcapitata.

In the actual environment, temperatures fluctuate drastically through season or global warming and are thought to affects risk of pollutants for aquatic biota; however, there is no report about the effect of water temperature on toxicity of widely used herbicide diuron to fresh water microalgae. The present research investigated inhibitory effect of diuron on growth and photosynthetic activity of a green alga Pseudokirchneriella subcapitata at five different temperatures (10, 15, 20, 25, and 30 °C) for 144 h of exposure. As a result, effective diuron concentrations at which a 50% decrease in algal growth occurred was increased with increasing water temperature ranging from 9.2 to 20.1 µg L(-1) for 72 h and 9.4-28.5 µg L(-1) for 144 h. The photochemical efficiency of photosystem II (F v/F m ratio) was significantly reduced at all temperatures by diuron exposure at 32 µg L(-1) after 72 h. Inhibition rates was significantly increased with decreased water temperature (P < 0.01). Intracellular H2O2 levels as an indicator of oxidative stress were also decreased with increasing temperature in both control and diuron treatment groups and were about 2.5 times higher in diuron treatment groups than that of controls (P < 0.01). Our results suggest water temperatures may affect the toxicokinetics of diuron in freshwater and should therefore be considered in environmental risk assessment.

Juvenile hormone activity of ethyl 4-(2-aryloxyhexyloxy)benzoates with precocious metamorphosis-inducing activity.

Ethyl 4-[2-(6-methyl-3-pyridyloxy)hexyloxy]benzoate (1) and ethyl 4-(2-phenoxyhexyloxy)benzoate (2), which induce precocious metamorphosis in larvae of Bombyx mori, a clear sign of juvenile hormone (JH) deficiency, showed JH activity when topically applied to allatectomized 4th instar larvae of B. mori. Compounds 1 and 2 induced precocious metamorphosis with doses at which they were effective as JH agonists.

Bleaching activity of 4-phenyl-3-(substituted benzylthio)-4H-1,2,4-triazoles.

A variety of 4-aryl- and 4-alkyl-3-(substituted benzylthio)-4H-1,2,4-triazoles were prepared and evaluated for their bleaching activity by the lettuce seedling test. Among the series of tested compounds, 4-(3-fluorophenyl)-3-(4-trifluoromethylbenzylthio)-4H-1,2,4-triazole (39) exhibited the highest bleaching activity, causing complete bleaching symptoms at 10 microM. In the dark condition, compound 39 inhibited the formation of such carotenoids as beta-carotene, violaxanthin, neoxanthin and lutein, resulting in the formation of zeta-carotene, phytoene, phytofluene and beta-zeacarotene, which were not detected in the untreated control. Treatment by compound 39 at 50 microM resulted in the amount of accumulated zeta-carotene being seven-fold higher than that of phytoene, phytofluene and beta-zeacarotene. These results suggest that compound 39 might have interfered with desaturation, especially zeta-carotene desaturation, during carotenoid biosynthesis.