Purification and characterization of a hydroxamic acid glucoside beta-glucosidase from wheat (Triticum aestivum L.) seedlings

A beta-glucosidase (EC 3.2.1.21) with a high affinity for cyclic hydroxamic acid beta-D-glucosides was purified from 48-h-old wheat (Triticum aestivum L.) seedlings. The activity occurred transiently at a high level during th e non-autotrophic stage of growth, and the nature of the transient occurren ce was correlated with that of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). The glucosidase had maximum activity at an acidic pH (pH 5.5) and the purified enzyme showed a high affinity for DIMBOA-Glc, V-max and K-m being 4100 nkat/mg protein and 0.27 mM, respectively. It also hydrolyzed p-nitrophenol beta-glycosides, as well as flavone and isoflavon e glucosides, but to a lesser extent. The results indicated that the primar y natural substrate for the glucosidase is DIMBOA-Glc and that the enzyme i s involved in defense against pathogens and herbivores in non-autotrophic w heat. The glucosidase was found to be present as oligomeric forms with a mo lecular mass of 260-300 kDa comprising 60- and 58-kDa monomers. The N-termi nal 12-amino-acid sequences of the two monomers were identical (Gly-Thr-Pro -(Ser?)-Lys-Pro-Ala-Glu-ProGly-Pro), and showed no similarity to those of o ther plant glucosidases. Polyacrylamide gel electrophoresis under nondenatu ring condition indicated the existence of at least eight isozymes. Three cu ltivars of Triticum aestivum had the same zone of glucosidase activity on z ymograms, but the activity zones of the Triticum species, T. aestivum L., T . spelta L. and T. turgidum? L.. had different mobilities.