Molecular cloning and characterization of a plant α1,3/4-fucosidase based on sequence tags from almond fucosidase I

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• 作者：Reinhard Zeleny ; Renaud Leonard ; Georg Dorfner ; Thomas Dalik ; Daniel Kolarich and Friedrich Altmann
• 关键词：Arabidopsis ; Fucosidase ; Lewis a ; Almond ; Plant glycosidase
• 刊名：Phytochemistry
• 出版年：2006
• 出版时间：April 2006
• 年：2006
• 卷：67
• 期：7
• 页码：641-648
• 全文大小：227 K

文摘

Our work with almond peptide N-glycosidase A made us interested also in the α1,3/4-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 α1,2-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 α1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The α1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.