Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Nov;8(11):2338–2346. doi: 10.1110/ps.8.11.2338

Mutations of endo-beta-N-acetylglucosaminidase H active site residueAs sp130 anG glu132: activities and conformations.

V Rao 1, T Cui 1, C Guan 1, P Van Roey 1
PMCID: PMC2144190  PMID: 10595536

Abstract

Endo-beta-N-acetylglucosaminidase H hydrolyzes the beta-(1-4)-glycosidic link of the N,N'-diacetylchitobiose core of high-mannose and hybrid asparagine-linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild-type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the beta-barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the beta-barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate-binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brünger A. T. Crystallographic refinement by simulated annealing. Application to a 2.8 A resolution structure of aspartate aminotransferase. J Mol Biol. 1988 Oct 5;203(3):803–816. doi: 10.1016/0022-2836(88)90211-2. [DOI] [PubMed] [Google Scholar]
  2. Brünger A. T., Kuriyan J., Karplus M. Crystallographic R factor refinement by molecular dynamics. Science. 1987 Jan 23;235(4787):458–460. doi: 10.1126/science.235.4787.458. [DOI] [PubMed] [Google Scholar]
  3. Davies G., Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure. 1995 Sep 15;3(9):853–859. doi: 10.1016/S0969-2126(01)00220-9. [DOI] [PubMed] [Google Scholar]
  4. Farber G. K., Petsko G. A. The evolution of alpha/beta barrel enzymes. Trends Biochem Sci. 1990 Jun;15(6):228–234. doi: 10.1016/0968-0004(90)90035-a. [DOI] [PubMed] [Google Scholar]
  5. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1991 Dec 1;280(Pt 2):309–316. doi: 10.1042/bj2800309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Henrissat B., Davies G. Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol. 1997 Oct;7(5):637–644. doi: 10.1016/s0959-440x(97)80072-3. [DOI] [PubMed] [Google Scholar]
  7. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  8. Lesk A. M., Brändén C. I., Chothia C. Structural principles of alpha/beta barrel proteins: the packing of the interior of the sheet. Proteins. 1989;5(2):139–148. doi: 10.1002/prot.340050208. [DOI] [PubMed] [Google Scholar]
  9. Maley F., Trimble R. B., Tarentino A. L., Plummer T. H., Jr Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem. 1989 Aug 1;180(2):195–204. doi: 10.1016/0003-2697(89)90115-2. [DOI] [PubMed] [Google Scholar]
  10. Matsumura I., Kirsch J. F. Is aspartate 52 essential for catalysis by chicken egg white lysozyme? The role of natural substrate-assisted hydrolysis. Biochemistry. 1996 Feb 13;35(6):1881–1889. doi: 10.1021/bi951671q. [DOI] [PubMed] [Google Scholar]
  11. McCarter J. D., Withers S. G. Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol. 1994 Dec;4(6):885–892. doi: 10.1016/0959-440x(94)90271-2. [DOI] [PubMed] [Google Scholar]
  12. O'Neill R. A. Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis. J Chromatogr A. 1996 Jan 12;720(1-2):201–215. doi: 10.1016/0021-9673(95)00502-1. [DOI] [PubMed] [Google Scholar]
  13. Raine A. R., Scrutton N. S., Mathews F. S. On the evolution of alternate core packing in eightfold beta/alpha-barrels. Protein Sci. 1994 Oct;3(10):1889–1892. doi: 10.1002/pro.5560031028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rao V., Guan C., Van Roey P. Crystal structure of endo-beta-N-acetylglucosaminidase H at 1.9 A resolution: active-site geometry and substrate recognition. Structure. 1995 May 15;3(5):449–457. doi: 10.1016/s0969-2126(01)00178-2. [DOI] [PubMed] [Google Scholar]
  15. Schmidt B. F., Ashizawa E., Jarnagin A. S., Lynn S., Noto G., Woodhouse L., Estell D. A., Lad P. Identification of two aspartates and a glutamate essential for the activity of endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus. Arch Biochem Biophys. 1994 Jun;311(2):350–353. doi: 10.1006/abbi.1994.1247. [DOI] [PubMed] [Google Scholar]
  16. Sergeev Y., Lee B. Alignment of beta-barrels in (beta/alpha)8 proteins using hydrogen-bonding pattern. J Mol Biol. 1994 Nov 25;244(2):168–182. doi: 10.1006/jmbi.1994.1717. [DOI] [PubMed] [Google Scholar]
  17. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
  18. Tarentino A. L., Plummer T. H., Jr Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods Enzymol. 1994;230:44–57. doi: 10.1016/0076-6879(94)30006-2. [DOI] [PubMed] [Google Scholar]
  19. Tarentino A. L., Plummer T. H., Jr, Maley F. The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H. J Biol Chem. 1974 Feb 10;249(3):818–824. [PubMed] [Google Scholar]
  20. Tarentino A. L., Quinones G., Changchien L. M., Plummer T. H., Jr Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3. Molecular cloning, primary sequence, and enzyme expression. J Biol Chem. 1993 May 5;268(13):9702–9708. [PubMed] [Google Scholar]
  21. Tarentino A. L., Quinones G., Schrader W. P., Changchien L. M., Plummer T. H., Jr Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H. J Biol Chem. 1992 Feb 25;267(6):3868–3872. [PubMed] [Google Scholar]
  22. Terwisscha van Scheltinga A. C., Armand S., Kalk K. H., Isogai A., Henrissat B., Dijkstra B. W. Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and X-ray structure of a complex with allosamidin: evidence for substrate assisted catalysis. Biochemistry. 1995 Dec 5;34(48):15619–15623. doi: 10.1021/bi00048a003. [DOI] [PubMed] [Google Scholar]
  23. Terwisscha van Scheltinga A. C., Hennig M., Dijkstra B. W. The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18. J Mol Biol. 1996 Sep 20;262(2):243–257. doi: 10.1006/jmbi.1996.0510. [DOI] [PubMed] [Google Scholar]
  24. Terwisscha van Scheltinga A. C., Kalk K. H., Beintema J. J., Dijkstra B. W. Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure. 1994 Dec 15;2(12):1181–1189. doi: 10.1016/s0969-2126(94)00120-0. [DOI] [PubMed] [Google Scholar]
  25. Trimble R. B., Maley F. Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H. Anal Biochem. 1984 Sep;141(2):515–522. doi: 10.1016/0003-2697(84)90080-0. [DOI] [PubMed] [Google Scholar]
  26. Van Roey P., Rao V., Plummer T. H., Jr, Tarentino A. L. Crystal structure of endo-beta-N-acetylglucosaminidase F1, an alpha/beta-barrel enzyme adapted for a complex substrate. Biochemistry. 1994 Nov 29;33(47):13989–13996. doi: 10.1021/bi00251a005. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES