Identification of Essential Residues of Human α-l-Fucosidase and Tests of Its Mechanism

Abstract

Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability of alpha-l-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their k(cat) values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Brønsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(lg) = -0.93, but that for k(cat) is biphasic, with beta(lg) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of beta(lg) for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter beta(lg) value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp(225) functions as a nucleophile and Glu(289) as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and (1)H NMR spectral analysis confirmed the formation of beta-fucosyl azide and alpha-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu(289), an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum

“…In addition, A. thaliana GH95 fucosidase (AtFuc95A) is inactive with pNP-Fuc or 1,3/4/6-linked fucosyl substrates with overall substrate specificity and an optimum pH of 5, similar to FgFCO1. As discussed above, both GH29 and GH95 fucosidases catalyze the specific hydrolysis of ␣-fucosyl linkages despite different active-site folds (22,23,(27)(28)(29) and opposite overall retention or inversion of the anomeric configuration during catalysis (27)(28)(29)(30)(31)(32). F. graminearum also encodes two hypothetical GH95 family enzymes, FG04734.1 and FG11516.1, based on a BLASTP search using AtFuc95A sequence (NP_195152.2) (18) as the query.…”

“…GH29 enzymes retain glycosidases, whereas GH95 enzymes are inverting glycosidases (27)(28)(29)(30)(31)(32). Enzymes in GH29 exist in all domains of life, whereas GH95 members have not been found in animals (15,26).…”

“…Human GH29 fucosidases, FUCA1 and FUCA2, are both active on ␣1,2/3/4/6-linked fucosyl substrates as well as pNP-Fuc (20,21,31,34). FUCA1 is a lysosomal enzyme whose deficiency causes fucosidosis, a disease characterized by progressive mental and motor deterioration (2).…”

“…However, the poor sequence conservation near the real catalytic Glu residues across different species has made it impractical to assign the key general acid/base Glu solely based on sequence alignment (15,23,27,31,32,36). Studies with the mechanism-based inhibitor 2-fluoro-fucosyl and analysis of site-directed mutants have confirmed the retaining catalytic mechanism (Fig.…”

Differences in the Substrate Specificities and Active-Site Structures of Two α-L-Fucosidases (Glycoside Hydrolase Family 29) fromBacteroides thetaiotaomicron

“…1A). 6,[8][9][10] These results suggest that GH29 can be divided into two subfamilies. One contains -L-fucosidases with relaxed substrate specificities that can act on para-nitorophenyl--Lfucopyranoside (pNP-Fuc) (referred to as GH29-A) (EC 3.2.1.51), whereas the members of the other subfamily specifically hydrolyze -(1!3/4)-fucosidic linkages and essentially do not act on pNP-Fuc (GH29-B) (EC 3.2.1.111).…”