Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase

The beta-glucosidase from Aspergillus niger (CMI. CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by revers e transcriptase-polymerase chain reaction, respectively. The cDNA was succe ssfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member o f glycosidase family 3. H-1-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enz yme hydrolyzes with net retention of anomeric configuration. Accordingly, t he enzyme was inactivated by a-deoxy-a-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K-I = 35.4 mM, through the trappi ng of a covalent glycosyl enzyme intermediate, The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamar in resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent ana lysis of high pressure liquid chromatography eluates by electrospray ioniza tion triple quadrupole mass spectrometry in the neutral loss mode allowed t he localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determinati on of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleoph ile within the sequence VMSDW, Asp-261 is fully conserved within this famil y, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with con duritol B epoxide.