Mechanistic and structural analysis of a family 31 alpha-glycosidase and its glycosyl-enzyme intermediate.
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-A 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.
Lovering
Andrew L
AL
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Lee
Seung Seo
SS
Kim
Young-Wan
YW
Withers
Stephen G
SG
Strynadka
Natalie C J
NC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D065089:Glycoside Hydrolase Inhibitors; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D017931:DNA Primers; D065089:Glycoside Hydrolase Inhibitors; D000520:alpha-Glucosidases