Background: Glutamate cysteine ligase (GCL) is a crucial enzyme involved in the synthesis of glutathione (GSH). Despite various studies on glutathione transferase and its essential role in detoxi cation and resistance to oxidative stress, GSH synthesis has not been described in silkworms to date. Silkworms form part of the lepidopterans that are considered as a model of agricultural pests. This study aimed to understand the GSH synthesis through the GCL in silkworms so that insecticides can be formulated to tackle agricultural pests.Methods and Results: Based on the amino acid sequence and phylogenetic tree, this GCL belonged to group 2 and designated bmGCL. Recombinant bmGCL was overproduced and puri ed to ensure homogeneity. Biochemical studies revealed that bmGCL uses ATP and Mg 2+ to ligate glutamate and cysteine, respectively. High expression levels of bmgcl mRNA and GSH were observed in the silkworm fat body after exposure to insecticides and UV-B. Moreover, we found an increase in bmgcl mRNA and GSH content during pupation in the silkworm fat body. Conclusion:In this study, we found GCL in the silkworm Bombyx mori and its biochemical properties were investigated. These observations indicate that bmGCL might play an important role in the resistance to oxidative stress in B. 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.

Background: Glutamate cysteine ligase (GCL) is a crucial enzyme involved in the synthesis of glutathione (GSH). Despite various studies on glutathione transferase and its essential role in detoxification and resistance to oxidative stress, GSH synthesis has not been described in silkworms to date. Silkworms form part of the lepidopterans that are considered as a model of agricultural pests. This study aimed to understand the GSH synthesis through the GCL in silkworms so that insecticides can be formulated to tackle agricultural pests. Methods and Results: Based on the amino acid sequence and phylogenetic tree, this GCL belonged to group 2 and designated bmGCL. Recombinant bmGCL was overproduced and purified to ensure homogeneity. Biochemical studies revealed that bmGCL uses ATP and Mg2+ to ligate glutamate and cysteine, respectively. High expression levels of bmgcl mRNA and GSH were observed in the silkworm fat body after exposure to insecticides and UV-B. Moreover, we found an increase in bmgcl mRNA and GSH content during pupation in the silkworm fat body. Conclusion: In this study, we found GCL in the silkworm Bombyx mori and its biochemical properties were investigated. These observations indicate that bmGCL might play an important role in the resistance to oxidative stress in B. mori.