Purification and characterization of UDP-glucose:cyclichydroxamic acid β-glucosyltransferases from maize seedlings

Abstract

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:cyclic hydroxamic acid β-glucosyltransferase (EC 2.4.1.-) activity, which was not detected in dry seeds, appeared in the embryo immediately after imbibition. The maximum activity was detected 24 and 12 h after germination in shoots and roots, respectively, and thereafter the activity gradually decreased to a low level. The increase and decrease in the enzyme activity preceded those of the DIMBOA glucoside, the major hydroxamic acid glucoside in maize, by 12 h. Purification of the enzyme from maize shoots was performed by ammonium sulfate precipitation, followed by chromatography on Blue Sepharose, Mono Q and Superdex 200. Two isozymes (GT1 and GT2) were observed on the chromatogram with Mono Q, and were separately purified to apparent homogeneity. The molecular masses of GT1 and GT2 were estimated to be 47 and 48 kDa, respectively, by SDS-PAGE analysis. Both isozymes glucosylated the cyclic hydroxamic acids, but they did not accept lactam species or flavonoids as 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 μM, and those of GT2 were 27.0 and 28.0 μM, respectively. The V max value of GT1 for DIMBOA was 14-fold greater than that for DIBOA, while the V max value of GT2 for DIMBOA was 2.2-fold greater than that for DIBOA.