Crystal structure of endo-beta-N-acetylglucosaminidase H at 1.9 A resolution: active-site geometry and substrate recognition.
BACKGROUND
Endo-beta-N-acetylglucosaminidase H (Endo H), an endoglycosidase secreted by Streptomyces plicatus, hydrolyzes the glycosidic bond between the core N-acetyglucosamine residues of asparagine-linked high-mannose oligosaccharides. Endo H is a commonly used reagent in glycobiology research, including the characterization of oligosaccharides in glycoproteins. On-going crystallographic studies of Endo H and related endoglycosidases are aimed at identifying the molecular features that determine the different substrate specificities of these enzymes.


RESULTS
The three-dimensional structure of Endo H has been determined to 1.9 A resolution. The overall fold of the enzyme is that of an irregular (alpha/beta)8-barrel comprising eight beta-strand/loop/alpha-helix units. Units 5 and 6 have very short loop sections at the top of the molecule and their alpha-helices are replaced by sections of extended geometry. The loop of unit 2 includes a small two-stranded antiparallel beta-sheet. A shallow curved cleft runs across the surface of the molecule from the area of units 5 and 6, over the core of the beta-barrel to the area of the beta-sheet of loop 2. This cleft contains the putative catalytic residues Asp130 and Glu132 above the core of the beta-barrel. These residues are surrounded by several aromatic residues. The loop 2 area of the cleft is formed by neutral polar residues, mostly asparagines.


CONCLUSIONS
The structure of Endo H is very similar to that of Endo F1, a closely related endoglycosidase secreted by Flavobacterium meningosepticum. Detailed comparison of the structures of Endo H and Endo F1 supports the model previously proposed for substate binding and recognition, in which the area of loop 2 determines the substrate specificity and the alpha-helices of units 5 and 6 are missing to accommodate the protein moiety of the substrate.
Rao
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V
Wadsworth Center, New York State Department of Health, Albany 12201-0509, USA.
Guan
C
C
Van Roey
P
P
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002240:Carbohydrate Sequence; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008351:Mannans / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011487:Protein Conformation; D013291:Streptococcus / Q000201:enzymology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008351:Mannans; D009844:Oligosaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase