Linked Life Data

10933800

Source:http://linkedlifedata.com/resource/pubmed/id/10933800

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
32
pubmed:dateCreated
2000-9-5
pubmed:databankReference
pubmed:abstractText
Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase clan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4', 6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2, 2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2, 6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
39
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
9826-36
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:10933800-Amino Acid Sequence, pubmed-meshheading:10933800-Base Sequence, pubmed-meshheading:10933800-Catalysis, pubmed-meshheading:10933800-Cloning, Molecular, pubmed-meshheading:10933800-Genes, Fungal, pubmed-meshheading:10933800-Glycoside Hydrolases, pubmed-meshheading:10933800-Glycosides, pubmed-meshheading:10933800-Indicators and Reagents, pubmed-meshheading:10933800-Mass Spectrometry, pubmed-meshheading:10933800-Models, Chemical, pubmed-meshheading:10933800-Molecular Sequence Data, pubmed-meshheading:10933800-Nitro Compounds, pubmed-meshheading:10933800-Phanerochaete, pubmed-meshheading:10933800-Sequence Analysis, DNA, pubmed-meshheading:10933800-Sequence Analysis, Protein, pubmed-meshheading:10933800-Sequence Homology, Amino Acid, pubmed-meshheading:10933800-alpha-Galactosidase
pubmed:year
2000
pubmed:articleTitle
Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase.
pubmed:affiliation
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't