Identification of an Arabidopsis gene encoding a GH95 alpha1,2-fucosidase active on xyloglucan oligo- and polysaccharides.
alpha1,2-linked fucose can be found on xyloglucans which are the main hemicellulose compounds of dicotyledons. The fucosylated nonasaccharide XXFG derived from xyloglucans plays a role in cell signaling and is active at nanomolar concentrations. The plant enzyme acting on this alpha1,2-linked fucose residues has been previously called fucosidase II; here we report on the molecular identification of a gene from Arabidopsis thaliana (At4g34260 hereby designed AtFuc95A) encoding this enzyme. Analysis of the predicted protein composed of 843 amino acids shows that the enzyme belongs to the glycoside hydrolase family 95 and has homologous sequences in different monocotyledons and dicotyledons. The enzyme was expressed recombinantly in Nicotiana bentamiana, a band was visible by Coomassie blue staining and its identity with the alpha1,2-fucosidase was assessed by an antibody raised against a peptide from this enzyme as well as by peptide-mass mapping. The recombinant AtFuc95A is active towards 2-fucosyllactose with a Km of 0.65 mM, a specific activity of 110 mU/mg and a pH optimum of 5 but does not cleave alpha1,3, alpha1,4 or alpha1,6-fucose containing oligosaccharides and p-nitrophenyl-fucose. The recombinant enzyme is able to convert the xyloglucan fragment XXFG to XXLG, and is also active against xyloglucan polymers with a Km value for fucose residues of 1.5mM and a specific activity of 36 mU/mg. It is proposed that the AtFuc95A gene has a role in xyloglucan metabolism.
Léonard
Renaud
R
Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria. renaud.leonard@boku.ac.at
Pabst
Martin
M
Bondili
Jayakumar Singh
JS
Chambat
Gérard
G
Veit
Christiane
C
Strasser
Richard
R
Altmann
Friedrich
F
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism*; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D005936:Glucans; D011994:Recombinant Proteins; D014990:Xylans; C029353:xyloglucan; C531251:GH95 alpha1,2-fucosidase protein, Arabidopsis; D005644:alpha-L-Fucosidase