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

Abstract

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.

Construction and analysis of the protein–protein interaction network for the olfactory system of the silkworm Bombyx mori

“…GSTs are multifunctional enzymes, and they have important biological characteristics and are involved in several processes. Most research GSTs has focused on the detoxification of toxic substances, such as pesticides (Ketterman et al, 2011; Yamamoto et al, 2018). GSTs also play an essential role in plant–insect interactions (Wang et al, 2017; Zou et al, 2016).…”

Structure and function of one unclassified-class glutathioneS-transferase inLeptinotarsa decemlineata

“…A NCBI blastp search with the LdGSTu1 sequence revealed that the highest identity matches with the PDB published unclassified GST, BmGSTu2 (PDB: 5ZFG) at a sequence identity of 60.43% [46,47,56]. In regards to insect GST classified classes (Delta, Epsilon, Omega, Sigma, Theta, and Zeta), LdGSTu1 exhibits the highest precent identities to Delta class, 40.38%, 39.62%, and 38.21% with AgGSTD 1-6 (PDB: 1PN9), AcGSTD 1-3 (PDB: 1JLV), and NlGSTD (PDB:…”

Investigation of the active site of an unclassified glutathione transferase in <i>Bombyx mori</i> by alanine scanning

“…We had previously revealed that these residues correspond with Ser67, Asn68, Asn102, Pro162, and Ser166 in bmGSTu2. 5) Among the five residues, we had characterized Glu66, Ser67, and Asn68 in our previous study. 12) In the structure of agGSTe2 (Anopheles gambiae epsilon-class GST) as well, the hydrogen-bonding network had been proposed.…”

“…The superposition of DmGSTE6 (PDB ID: 4YH2) with bmGSTu2 revealed that the corresponding motifs in bmGSTu2 were Ser67, Asn68, and Leu99. We had previously reported that Leu99 of bmGSTu2 forms part of the lock-and-key motif, which is crucial for stabilizing the hydrophobic interactions of GST monomers, 5) while Ser67 and Asn68 of bmGSTu2 are GSH-site residues. 12) We could not find any motif similar to the epsilon clasp motif in bmGSTu2.…”

“…[3][4][5][6][7][8][9][10] Recently, X-ray structures of delta-class (bmGSTD), sigma-class (bmGSTS), omega-class (bmGSTO), unclassified (bmGSTu), and unclassified 2 (bmGSTu2) GSTs in B. mori have been determined. 5,6,8,9,11) We have reported bmGSTu2 (Protein Data Bank (PDB) ID: 5ZFG) to catalyze diazinon metabolism, thus explaining its role in the insecticide resistance of B. mori. To improve our understanding of the molecular basis of bmGSTu2 catalysis, we have examined the structure and catalytic function of bmGSTu2.…”