Purification and characterization of UDP-glucose : cyclic hydroxamic acid beta-glucosyltransferases from maize seedlings

The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy analogue (DIMBOA), accumulate as glucosides in a high amount in maize (Zea mays) during the juvenile stage of growth. UDP-glucose:cycli c hydroxamic acid beta -glucosyltransferase (EC 2.4.1.-) activity, which wa s not detected in dry seeds, appeared in the embryo immediately after imbib ition. The maximum activity was detected 24 and 12 h after germination in s hoots and roots, respectively, and thereafter the activity gradually decrea sed to a low level. The increase and decrease in the enzyme activity preced ed those of the DIMBOA glucoside, the major hydroxamic acid glucoside in ma ize, by 12 h. Purification of the enzyme from maize shoots was performed by ammonium sulfate precipitation, followed by chromatography on Blue Sepharo se, Mono Q and Superdex 200. Two isozymes (GT1 and GT2) were observed on th e chromatogram with Mono Q, and were separately purified to apparent homoge neity. The molecular masses of GT1 and GT2 were estimated to be 47 and 48 k Da, respectively, by SDS-PAGE analysis. Both isozymes glucosylated the cycl ic hydroxamic acids, but they did not accept lactam species or flavonoids a s substrates. The substrate specificity of the two isozymes for the cyclic hydroxamic acids was different, GT1 being more specific for DIMBOA than GT2 . The K-m values of GT1 for DIMBOA and DIBOA were 24.4 and 191 muM, and tho se of GT2 were 27.0 and 28.0 muM, respectively. The V-max value of GT1 for DIMBOA was 14-fold greater than that for DIBOA, while the V-max value of GT 2 for DIMBOA was 2.2-fold greater than that for DIBOA. (C) 2001 Editions sc ientifiques et medicales Elsevier SAS.