| Autor: |
Dan, Siegel, Marton, Ira, Dekel, Mara, Bravdo, Ben-Ami, He, Shouming, Withers, Stephen G., Shoseyov, Oded |
| Zdroj: |
Journal of Biological Chemistry; February 2000, Vol. 275 Issue: 7 p4973-4980, 8p |
| Abstrakt: |
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 cerevisiaeandPichia 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 wentiienzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem.355, 438–442). |
| Databáze: |
Supplemental Index |
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