Cloning, Expression, Characterization, and Nucleophile Identification of Family 3, Aspergillus nigerβ-Glucosidase*
By S. Dan, I. Marton, M. Dekel, B. Bravdo, Shouming He, S. Withers, O. Shoseyov. Journal of Biological Chemistry, 2000
148 citations recorded by the source corpus.
The β-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 reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae andPichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3.1H-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by 2-deoxy-2-fluoro β-glucosyl fluoride, with kinetic parameters of ki = 4.5 min−1, KI = 35.4 mm, through the trapping of a covalent glycosyl enzyme intermediate. The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW. Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in theAspergillus wentii enzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 438–442).
Subfields: Biomedical Engineering, Biotechnology, Organic Chemistry
View Paper: https://www.semanticscholar.org/paper/a4b620c1b08b8d8f3e8a64ba24ef161b5b2cca0e
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Citations in the corpus
- Microbial β-Glucosidase: Sources, Production and Applications (2017, background): “The nucleophile of β-glucosidase belonging to GH 3 has been identified as Asp residue which is highly conserved; while catalytic general acid/base residue appeared to be presented by different motifs in different members, although mostly it is His-Asp dyad motif in which the histidine side chain inv”
- Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives (2009, background): “BGLs from both families hydrolyze β-1,4-glycosidic bonds using the retaining mechanism [65].”
- Microbial β-Glucosidases: Cloning, Properties, and Applications (2002, background): “…such as Trichoderma reesei (Barnett et al., 1991), Aspergillus sp. (Takashima et al., 1999), S. cerevisiae (Dan et al., 2000), and Pichia pastoris (Dan et al., 2000), with the exception of β-glucosidase from Talaromyces emersonii (Morrison et al., 1990) and Phanerochaete chrysosporium (Li and…”
- Microbial cellulolytic enzymes: diversity and biotechnology with reference to lignocellulosic biomass degradation (2020, background): “This reaction occurs via double-displacement mechanism (Dan et al. 2000). b-glucosidases are responsible for the final step in cellulose hydrolysis.”
- Fungal enzyme sets for plant polysaccharide degradation (2011, unknown): “GH3 An18g03570 (bgl1), An07g09760, An08g08240, An11g00200, An14g01770, An17g00520, An03g05330, An06g02040, An15g01890, An11g06090, An15g04800 (Dan et al. 2000; Pel et al. 2007)”