title: Bifunctional family 3 glycoside hydrolases from barley with alpha -L-arabinofuranosidase and beta -D-xylosidase activity. Characterization, primary structures, and COOH-terminal processing.
abstract: An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4'-nitrophenyl alpha-l-arabinofuranoside is only slightly higher than during hydrolysis of 4'-nitrophenyl beta-d-xyloside. Thus, the enzyme is actually a bifunctional alpha-l-arabinofuranosidase/beta-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4'-nitrophenyl beta-d-xyloside, with a catalytic efficiency approximately 30-fold higher than with 4'-nitrophenyl alpha-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 --> 4)-beta-d-xylan endohydrolase. Both enzymes hydrolyze (1 --> 4)-beta-d-xylopentaose, and ARA-I can also degrade (1 --> 5)-alpha-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues.
authors.affiliation: Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Lee
authors.forename: Maria
authors.initials: M
authors.lastname: Hrmova
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Burton
authors.forename: Jelle
authors.initials: J
authors.lastname: Lahnstein
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: D000595:Amino Acid Sequence; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D013379:Substrate Specificity; D014995:Xylosidases / Q000032:analysis / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase