@article { ISI:000452746700002,
	title = {Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata},
	journal = {GLYCOBIOLOGY},
	volume = {28},
	number = {12},
	year = {2018},
	month = {DEC},
	pages = {925-932},
	abstract = {alpha-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 oc-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 oc-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 alpha-L-fucosidase from the spider Nephilingis cruentata (NcFuc) with a K-m value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2{{\textquoteright}{\textquoteright}}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(cat) of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower k(cat) value of E59A. This was supported by the 57-fold increase in the k(cat) of 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.},
	issn = {0959-6658},
	doi = {10.1093/glycob/cwy083},
	author = {Perrella, Natalia N. and Withers, Stephen G. and Lopes, Adriana R.}
}
