Probing the catalytically essential residues of the alpha-L-fucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus.
Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.
Cobucci-Ponzano
Beatrice
B
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131, Naples, Italy.
Mazzone
Marialuisa
M
Rossi
Mosè
M
Moracci
Marco
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics; D019766:Genes, Archaeal; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019641:DNA, Archaeal; D005644:alpha-L-Fucosidase