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Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata.
- Source :
-
Glycobiology . Dec2018, Vol. 28 Issue 12, p925-932. 8p. - Publication Year :
- 2018
-
Abstract
- α- l -Fucosidases are widely occurring enzymes that remove fucose residues from N - and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α- l -fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α- l -fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α- l -fucosidase from the spider Nephilingis cruentata (NcFuc) with a K mvalue for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto- N -difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower k catof D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower k catvalue of E59A. This was supported by the 57-fold increase in the k catof E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases. [ABSTRACT FROM AUTHOR]
- Subjects :
- *FUCOSIDASES
*GLYCOSIDASES
*FUCOSE
*SPIDERS
*AMINO acid sequence
Subjects
Details
- Language :
- English
- ISSN :
- 09596658
- Volume :
- 28
- Issue :
- 12
- Database :
- Academic Search Index
- Journal :
- Glycobiology
- Publication Type :
- Academic Journal
- Accession number :
- 133186957
- Full Text :
- https://doi.org/10.1093/glycob/cwy083