title: Family 39 alpha-l-iduronidases and beta-D-xylosidases react through similar glycosyl-enzyme intermediates: identification of the human iduronidase nucleophile.
abstract: The inclusion of both beta-D-xylosidases and alpha-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 alpha-L-iduronidase reveals that, as with the beta-D-xylosidases, alpha-L-iduronidase is a retaining glycosidase. Using two different mechanism-based inactivators, 5-fluoro-alpha-L-iduronyl fluoride and 2-deoxy-2-fluoro-alpha-L-iduronyl fluoride, the active site nucleophile in the human alpha-L-iduronidase was identified as Glu299 within the (295)IYNDEAD(301) sequence. The equivalent, though loosely predicted, glutamic acid was identified as the nucleophile in the family 39 beta-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 (2,5)B conformation is predicted.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Catharine E
authors.initials: CE
authors.lastname: Nieman
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Wong
authors.forename: Shouming
authors.initials: S
authors.lastname: He
authors.forename: Lorne
authors.initials: L
authors.lastname: Clarke
authors.forename: John J
authors.initials: JJ
authors.lastname: Hopwood
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007067:Iduronic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D007068:Iduronidase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D007067:Iduronic Acid; D018698:Glutamic Acid; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; D007068:Iduronidase