Family 39 α-L-iduronidases and β-D-xylosidases react through similar glycosyl-enzyme intermediates: Identification of the human iduronidase nucleophile

Catharine E. Nieman, Alexander W. Wong, Shouming He, Lorne Clarke, John J. Hopwood, Stephen G. Withers

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

Abstract

The inclusion of both β-D-xylosidases and α-L-iduronidases within the same sequence-related family (family 39), despite the considerable difference in substrate structures and poor sequence conservation around the putative nucleophile, raises concerns about whether a common mechanism is followed by the two enzymes. A novel anchimeric assistance mechanism for iduronidases involving a lactone intermediate is one possibility. NMR analysis of the methanolysis reaction catalyzed by human α-L-iduronidase reveals that, as with the β-D-xylosidases, α-L-iduronidase is a retaining glycosidase. Using two different mechanism-based inactivators, 5-fluoro-α-L-iduronyl fluoride and 2-deoxy-2-fluoro-α-L-iduronyl fluoride, the active site nucleophile in the human α-L-iduronidase was identified as Glu299 within the 295IYNDEAD301 sequence. The equivalent, though loosely predicted, glutamic acid was identified as the nucleophile in the family 39 β-D-xylosidase from Bacillus sp. [Vocadlo, D., et al. (1998) Biochem. J. 335, 449-455]; thus, a common mechanism involving a covalent glycosyl-enzyme intermediate that adopts the rather uncommon 2.5B conformation is predicted.

Original languageEnglish
Pages (from-to)8054-8065
Number of pages12
JournalBiochemistry
Volume42
Issue number26
DOIs
Publication statusPublished or Issued - 8 Jul 2003

ASJC Scopus subject areas

  • Biochemistry

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