@article{73983ce091fe4df2bfee779f18557660,
title = "Crystal structure of native β-N-acetylhexosaminidase isolated from Aspergillus oryzae sheds light onto its substrate specificity, high stability, and regulation by propeptide",
abstract = "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 {\AA} 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. Database: Structural data are available in the PDB database under the accession number 5OAR. Enzyme: β-N-acetylhexosaminidase (EC 3.2.1.52).",
keywords = "X-ray crystallography, active pocket, carbohydrate biotechnology, glycosylation, mass spectrometry, propeptide",
author = "Jana {\v S}kerlov{\'a} and Jan Bl{\'a}ha and Petr Pachl and Kate{\v r}ina Hofbauerov{\'a} and Zden{\v e}k Kuka{\v c}ka and Petr Man and Petr Pompach and Petr Nov{\'a}k and Zbyszek Otwinowski and Ji{\v r}{\'i} Brynda and Ond{\v r}ej Van{\v e}k and Pavl{\'i}na {\v R}ez{\'a}{\v c}ov{\'a}",
note = "Funding Information: The authors thank V{\'a}clav Veverka and Hillary Hoffman for critical proofreading of the manuscript. JS thanks Monika Oweyssi for personal help and support during the preparation of the manuscript. This work was supported by BIOCEV (ERDF CZ.1.05/1.1.00/ 02.0109 and CZ.02.1.01/0.0/0.0/16_013/0001776), the Ministry of Education, Youth and Sport of the Czech Republic (programs {\textquoteleft}NPU I{\textquoteright}, projects LO1304 and LO1509; {\textquoteleft}NPU II{\textquoteright}, project LQ1604; projects LG14009, LH15010, and LM2015043 CIISB for CMS BIOCEV; project LTC17065 in frame of the COST Action CA15126; and project LD15089 in frame of the COST Action BM1403), the Czech Science Foundation (15-05677S, 16-24309S, and 15-15181S) and in part by the Czech Academy of Sciences (RVO 68378050, 61388971, and 61388963) and by the Charles University (UNCE 204025/2012 and SVV 260079/2014). The use of beamline MX14.2 operated by the Helmholtz– Zentrum Berlin at the BESSY II electron storage ring (Berlin-Adlershof, Germany) to collect diffraction data is gratefully acknowledged. Funding Information: The authors thank V?clav Veverka and Hillary Hoffman for critical proofreading of the manuscript. JS thanks Monika Oweyssi for personal help and support during the preparation of the manuscript. This work was supported by BIOCEV (ERDF CZ.1.05/1.1.00/02.0109 and CZ.02.1.01/0.0/0.0/16_013/0001776), the Ministry of Education, Youth and Sport of the Czech Republic (programs ?NPU I?, projects LO1304 and LO1509; ?NPU II?, project LQ1604; projects LG14009, LH15010, and LM2015043 CIISB for CMS BIOCEV; project LTC17065 in frame of the COST Action CA15126; and project LD15089 in frame of the COST Action BM1403), the Czech Science Foundation (15-05677S, 16-24309S, and 15-15181S) and in part by the Czech Academy of Sciences (RVO 68378050, 61388971, and 61388963) and by the Charles University (UNCE 204025/2012 and SVV 260079/2014). The use of beamline MX14.2 operated by the Helmholtz?Zentrum Berlin at the BESSY II electron storage ring (Berlin-Adlershof, Germany) to collect diffraction data is gratefully acknowledged. Publisher Copyright: {\textcopyright} 2017 Federation of European Biochemical Societies",
year = "2018",
month = feb,
doi = "10.1111/febs.14360",
language = "English (US)",
volume = "285",
pages = "580--598",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "3",
}