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Structure and Activity of the Streptomyces coelicolor A3(2) β-N-Acetylhexosaminidase Provides Further Insight into GH20 Family Catalysis and Inhibition

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INRS-Institut Armand-Frappier, Université du Québec, 531 Boul. des Prairies, Laval, Québec H7V 1B7, Canada
PROTEO, the Québec Network for Research on Protein Function, Structure, and Engineering, 1045 Avenue de la Médecine, Université Laval, Québec, Québec G1V 0A6, Canada
§ GRASP, the Groupe de Recherche Axé sur la Structure des Protéines, 3649 Promenade Sir William Osler, McGill University, Montréal, Québec H3G 0B1, Canada
Military Institute of Science and Technology, 17 Hoang Sam, Hanoi, Vietnam
Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam
Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom
*E-mail: [email protected]. Fax: (450) 686-5501. Tel.: (450) 687-5010, ext. 4212.
Cite this: Biochemistry 2014, 53, 11, 1789–1800
Publication Date (Web):February 21, 2014
Copyright © 2014 American Chemical Society
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Abstract Image

β-N-acetylhexosaminidases (HEX) are glycosidases that catalyze the glycosidic linkage hydrolysis of gluco- and galacto-configured N-acetyl-β-d-hexosaminides. These enzymes are important in human physiology and are candidates for the biocatalytic production of carbohydrates and glycomimetics. In this study, the three-dimensional structure of the wild-type and catalytically impaired E302Q HEX variant from the soil bacterium Streptomyces coelicolor A3(2) (ScHEX) were solved in ligand-free forms and in the presence of 6-acetamido-6-deoxy-castanospermine (6-Ac-Cas). The E302Q variant was also trapped as an intermediate with oxazoline bound to the active center. Crystallographic evidence highlights structural variations in the loop 3 environment, suggesting conformational heterogeneity for important active-site residues of this GH20 family member. The enzyme was investigated for its β-N-acetylhexosaminidase activity toward chitooligomers and pNP-acetyl gluco- and galacto-configured N-acetyl hexosaminides. Kinetic analyses confirm the β(1–4) glycosidic linkage substrate preference, and HPLC profiles support an exoglycosidase mechanism, where the enzyme cleaves sugars from the nonreducing end of substrates. ScHEX possesses significant activity toward chitooligosaccharides of varying degrees of polymerization, and the final hydrolytic reaction yielded pure GlcNAc without any byproduct, promising high applicability for the enzymatic production of this highly valued chemical. Thermostability and activation assays further suggest efficient conditions applicable to the enzymatic production of GlcNAc from chitooligomers.

Supporting Information

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A sequence alignment of ScHEX with members of the GH20 β-N-acetylhexosaminidase family and Michaelis–Menten kinetics profiles for the hydrolysis of (GlcNAc)2–6 by ScHEX. This material is available free of charge via the Internet at

Accession Codes

Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4C7D, 4C7F, and 4C7G.

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This article is cited by 21 publications.

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  11. Xi Chen, Mengyu Li, Yongzhong Wang, Rupei Tang, Min Zhang. Biochemical characteristics and crystallographic evidence for substrate-assisted catalysis of a β-N-acetylhexosaminidase in Akkermansia muciniphila. Biochemical and Biophysical Research Communications 2019, 517 (1) , 29-35.
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  14. Joan Coines, Mercedes Alfonso‐Prieto, Xevi Biarnés, Antoni Planas, Carme Rovira. Oxazoline or Oxazolinium Ion? The Protonation State and Conformation of the Reaction Intermediate of Chitinase Enzymes Revisited. Chemistry – A European Journal 2018, 24 (72) , 19258-19265.
  15. Piyanat Meekrathok, Keith A. Stubbs, Wipa Suginta. Potent inhibition of a GH20 exo-β-N-acetylglucosaminidase from marine Vibrio bacteria by reaction intermediate analogues. International Journal of Biological Macromolecules 2018, 115 , 1165-1173.
  16. Tian Liu, Yanwei Duan, Qing Yang. Revisiting glycoside hydrolase family 20 β-N-acetyl-d-hexosaminidases: Crystal structures, physiological substrates and specific inhibitors. Biotechnology Advances 2018, 36 (4) , 1127-1138.
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  19. Nhung Nguyen-Thi, Nicolas Doucet. Combining chitinase C and N-acetylhexosaminidase from Streptomyces coelicolor A3(2) provides an efficient way to synthesize N-acetylglucosamine from crystalline chitin. Journal of Biotechnology 2016, 220 , 25-32.
  20. Gonzalo N. Bidart, Jesús Rodríguez-Díaz, María J. Yebra, . The Extracellular Wall-Bound β- N -Acetylglucosaminidase from Lactobacillus casei Is Involved in the Metabolism of the Human Milk Oligosaccharide Lacto- N -Triose. Applied and Environmental Microbiology 2016, 82 (2) , 570-577.
  21. Cristina Val-Cid, Xevi Biarnés, Magda Faijes, Antoni Planas, . Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases. PLOS ONE 2015, 10 (5) , e0128075.

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