β-N-acetylhexosaminidase from the fungus Aspergillus oryzae is a secreted extracellular enzyme that cleaves chitobiose into constituent monosaccharides. It belongs to the GH 20 glycoside hydrolase family and consists of two N-glycosylated catalytic cores noncovalently associated with two 10-kDa O-glycosylated propeptides. We used X-ray diffraction and mass spectrometry to determine the structure of A. oryzae β-N-acetylhexosaminidase isolated from its natural source. The three-dimensional structure determined and refined to a resolution of 2.3 Å revealed that this enzyme is active as a uniquely tight dimeric assembly further stabilized by N- and O-glycosylation. The propeptide from one subunit forms extensive noncovalent interactions with the catalytic core of the second subunit in the dimer, and this chain swap suggests the distinctive structural mechanism of the enzyme's activation. Unique structural features of β-N-acetylhexosaminidase from A. oryzae define a very stable and robust framework suitable for biotechnological applications. The crystal structure reported here provides structural insights into the enzyme architecture as well as the detailed configuration of the active site. These insights can be applied to rational enzyme engineering.
mass spectrometry, glycosylation, X-ray crystallography, active pocket, carbohydrate biotechnology, propeptide
NCBI PubMed ID: 29239122Publication DOI: 10.1111/febs.14360Journal NLM ID: 101229646Publisher: Blackwell Publishing
Correspondence: Vaněk O
; Řezáčová P
Institutions: Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, Prague, Czech Republic, Institute of Molecular Genetics, The Czech Academy of Sciences, Prague, Czech Republic, Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic, Institute of Microbiology, The Czech Academy of Sciences, Prague, Czech Republic, Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic, UT Southwestern Medical Center, Dallas, USA
Methods: gel filtration, SDS-PAGE, MS/MS, LC-MS, reversed-phase chromatography, precipitation, x-ray, FTIR-MS
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