Molecular cloning and characterization of a plant alpha 1,3/4-fucosidase based on sequence tags from almond fucosidase I

Our work with almond peptide N-glycosidase A made us interested also in the alpha 1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion e...

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Published in:Phytochemistry (Oxford) 2006-04, Vol.67 (7), p.641-648
Main Authors: Zeleny, Reinhard, Leonard, Renaud, Dorfner, Georg, Dalik, Thomas, Kolarich, Daniel, Altmann, Friedrich
Format: Article
Language:English
Subjects:
Arabidopsis thaliana
Oryza sativa
Pichia pastoris
Prunus dulcis
Streptomyces
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Summary:Our work with almond peptide N-glycosidase A made us interested also in the alpha 1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha 1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha 1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha 1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.
ISSN:0031-9422
DOI:10.1016/j.phytochem.2006.01.021