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Mechanistic and structural analysis of a family 31 alpha-glycosidase and its glycosyl-enzyme intermediate

TitleMechanistic and structural analysis of a family 31 alpha-glycosidase and its glycosyl-enzyme intermediate
Publication TypeJournal Article
Year of Publication2005
AuthorsLovering, AL, Lee, SS, Kim, YW, Withers, SG, Strynadka, NCJ
JournalJOURNAL OF BIOLOGICAL CHEMISTRY
Volume280
Pagination2105-2115
Date PublishedJAN 21
ISSN0021-9258
Abstract

We have determined the first structure of a family 31 alpha-glycosidase, that of YicI from Escherichia coli, both free and trapped as a 5-fluoroxylopyranosyl-enzyme intermediate via reaction with 5-fluoro-alpha-D-xylopyranosyl fluoride. Our 2.2- Angstrom resolution structure shows an intimately associated hexamer with structural elements from several monomers converging at each of the six active sites. Our kinetic and mass spectrometry analyses verified several of the features observed in our structural data, including a covalent linkage from the carboxylate side chain of the identified nucleophile Asp(416) to C-1 of the sugar ring. Structure-based sequence comparison of YicI with the mammalian alpha-glucosidases lysosomal alpha-glucosidase and sucrase-isomaltase predicts a high level of structural similarity and provides a foundation for understanding the various mutations of these enzymes that elicit human disease.

DOI10.1074/jbc.M410468200