NDLI: Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases* S⃞

Content Provider	PubMed Central
Author	Abbott, D. Wade Macauley, Matthew S. Vocadlo, David J. Boraston, Alisdair B.
Abstract	Endo-β-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (Kd = 28 μm) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.
Related Links	http://dx.doi.org/10.1074/jbc.m809663200
Ending Page	11689
Page Count	14
Starting Page	11676
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	17
Volume Number	284
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2009-04-24
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases

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