Expression and characterization of a sigma-class glutathione S-transferase of the fall webworm, Hyphantria cunea.
A cDNA encoding glutathione S-transferase (GST) of the fall webworm, Hyphantria cunea, was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone (hcGST) was sequenced and deduced for amino acid sequence, which revealed 87, 59, and 42% identities to Sigma-class GSTs from Bombyx mori, Manduca sexta, and Blattella germanica respectively. A recombinant hcGST protein (rhcGST) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rhcGST retained more than 75% of its original GST activity after incubation at pHs 6 to 11. Incubation for 30 min at temperatures below 50 degrees C scarcely affected the activity. rhcGST was able to catalyze the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. We also found that as compared to B. mori Sigma-class GST, rhcGST had a higher affinity for fenitrothion, an organophosphorus insecticide.
Yamamoto
Kohji
K
Institute of Genetic Resources, Faculty of Agriculture, Kyushu University Graduate School, Fukuoka, Japan. yamamok@agr.kyushu-u.ac.jp
Fujii
Hiroshi
H
Aso
Yoichi
Y
Banno
Yutaka
Y
Koga
Katsumi
K
D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000494:pharmacology; D007814:Larva / Q000378:metabolism; D015227:Lipid Peroxidation / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D009036:Moths / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature
D000447:Aldehydes; D018076:DNA, Complementary; D004137:Dinitrochlorobenzene; D007306:Insecticides; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C027576:4-hydroxy-2-nonenal