Avenacosidase from oat: purification, sequence analysis and biochemical characterization of a new member of the BGA family of beta-glucosidases.
A protein consisting of 60 kDa subunits (As-P60) was isolated from etiolated oat seedlings (Avena sativa L.) and characterized as avenacosidase, a beta-glucosidase that belongs to a preformed defence system of oat against fungal infection. The enzyme is highly aggregated; it consists of 300-350 kDa aggregates and multimers thereof. Dissociation by freezing/thawing leads to complete loss of enzyme activity. The specificity of the enzyme was investigated with para-nitrophenyl derivatives which serve as substrates, in decreasing order beta-fucoside, beta-glucoside, beta-galactoside, beta-xyloside. The corresponding orthonitrophenyl glycosides are less well accepted. No hydrolysis was found with alpha-glycosides and beta-thioglucoside. An anti-As-P60 antiserum was prepared and used for isolation of a cDNA clone coding for As-P60. A presequence of 55 amino acid residues was deduced from comparison of the cDNA sequence with the N-terminal sequence determined by Edman degradation of the mature protein. The presequence has the characteristics of a stroma-directing signal peptide; localization of As-P60 in plastids of oat seedlings was confirmed by western blotting. The amino acid sequence revealed significant homology (> 39% sequence identity) to beta-glucosidases that are constituents of a defence mechanism in dicotyledonous plants. 34% sequence identity was even found with mammalian and bacterial beta-glucosidases of the BGA family. Avenacosidase extends the occurrence of this family of beta-glucosidases to monocotyledonous plants.
Gus-Mayer
S
S
Botanisches Institut, Universität München, Germany.
Brunner
H
H
Schneider-Poetsch
H A
HA
Rüdiger
W
W
D000595:Amino Acid Sequence; D018554:Avena / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002451:Cell Compartmentation; D017343:Genes, Plant / Q000235:genetics; D006027:Glycosides / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D018087:Plastids / Q000201:enzymology; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000145:classification* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D006027:Glycosides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C087992:avenacosidase; D001617:beta-Glucosidase