Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase.
The baculovirus ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, Autographa californica nucleopolyhedrovirus EGT has been overproduced and purified, and its kinetic properties determined. The enzyme was purified 1100-fold to near-homogeneity using only two major steps, ion-exchange and gel-filtration chromatography. EGT activity was eluted from the gel-filtration column as a single peak corresponding to a 260+/-50 kDa protein, suggesting that the enzyme is an oligomer of three to five subunits, as the subunit molecular mass is approximately 56 kDa. Kinetic analysis showed that EGT has broadly similar specificities for UDP-galactose and UDP-glucose (kcat/Km=1790.8 and 902.1 respectively) when ecdysone is used as the other substrate. On the other hand, it shows marked differences in specificity for the various ecdysteroids tested. Ecdysone seems to be the optimal substrate (kcat/Km=7101.1), whereas 3-dehydroecdysone, an ecdysone precursor in Lepidoptera, is seven times less favourable (kcat/Km=1085.7). Notably, 20-hydroxyecdysone, the active form of the hormone, is conjugated very poorly (kcat/Km=31.6). Analysis of the data revealed that the enzyme mechanism involves the formation of an ecdysteroid-UDP-sugar-enzyme ternary complex. This work represents the most detailed biochemical characterization of an EGT to date.
Evans
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Department of Biology, Imperial College of Science, Technology, and Medicine, Prince Consort Road, London SW7 2BB, U.K.
O'reilly
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D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D026461:Ecdysteroids; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007915:Lepidoptera / Q000502:physiology / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013256:Steroids / Q000378:metabolism; D013379:Substrate Specificity
D026461:Ecdysteroids; D011994:Recombinant Proteins; D013256:Steroids; D005964:Glucosyltransferases; C086659:ecdysteroid UDP-glucosyltransferase