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 the non-autotrophic stage of growth, and the nature of the transient occurrence 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, Vmax and Km being 4100 nkat/mg protein and 0.27 mM, respectively. It also hydrolyzed p-nitrophenol beta-glycosides, as well as flavone and isoflavone glucosides, but to a lesser extent. The results indicated that the primary natural substrate for the glucosidase is DIMBOA-Glc and that the enzyme is involved in defense against pathogens and herbivores in non-autotrophic wheat. The glucosidase was found to be present as oligomeric forms with a molecular mass of 260-300 kDa comprising 60- and 58-kDa monomers. The N-terminal 12-amino-acid sequences of the two monomers were identical (Gly-Thr-Pro-(Ser?)-Lys-Pro-Ala-Glu-Pro-Ile-Gly-Pro), and showed no similarity to those of other plant glucosidases. Polyacrylamide gel electrophoresis under nondenaturing condition indicated the existence of at least eight isozymes. Three cultivars of Triticum aestivum had the same zone of glucosidase activity on zymograms, but the activity zones of the Triticum species, T. aestivum L., T. spelta L. and T. turgidum L., had different mobilities.
Sue
M
M
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan. sue@kais.kyoto-u.ac.jp
Ishihara
A
A
Iwamura
H
H
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000502:physiology; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D010446:Peptide Fragments; C406791:2,4-dihydroxy - 7-methoxy-1,4-benzoxazin-3-one glucoside beta-glucosidase; D001617:beta-Glucosidase