P.Man1,2, P.Pompach1,2, V.Havlíček1, O.Plíhal1,2, J.Sklenář1,2, V.Křen1 and K.Bezouška1,2


1Institute of Microbiology, Academy of Sciences of Czech Republic, 14220 Praha 4, 2Department of Biochemistry, Faculty of Science, Charles University Prague, 12840 Praha 2


Fungal β-N-acetylhexosaminidases (chitobiases, EC are secreted enzymes that hydrolyze chitobiose into monosaccharides. The enzymes are physiologically important for the formation of septa in molds, germ tubes in yeast, and fruit bodies in fungi [1]. Moreover, they have found many applications in biotechnologies due to their unique abilities to synthesize new and unusual oligosaccharide structures [2].

We have cloned and sequenced β-N-acetylhexosaminidase from the collection strain of Aspergillus oryzae CCF1066 [3]. This enzyme is composed of four polypeptide segments. The N-terminal signal peptide is cleaved intracellularly. The zincin-like and catalytical domain define the enzyme as a member of family 20 glycohydrolases. Homology modeling revealed significant similarity with the two crystallized bacterial β-N-acetylhexosaminidases.

Fungal β-N-acetylhexosaminidases contain a unique N-terminal propeptide that is processed intracellularly before the secretion of the enzyme. Detailed pulse-chase and inhibition studies revealed that the propeptide is processed very early during the biosynthesis, just after the addition of N-glycans. The propeptide must be processed in order to asssist in enzyme refolding, activation, and dimerization. Monomeric enzyme subunits devoid of the propeptide are inactive, cannot dimerize, and may not be secreted from the cell [4]. Dimers containing a single propeptide are secreted at only half the rate of those containing both propeptides, and have lower specific activity [5].

The unique propeptide properties are undoubtedly dictated by its unusual structural features. Therefore we turned our attention toward the characterization of its primary structure. The propeptide has a prolin rich C-terminal part with several potential O-glycosylation sites. The glycosylation pattern is extremely difficult to solve as there are at least two or three glycans attached to serin or threonin. Moreover, the glycosyaltion appeared rather resistant to chemical cleavage and it also protected the C-terminal part of the propeptide from proteolysis. Partial primary structure including glycosylation was solved by tandem mass spectrometry, namely collision induced dissociation (CID). Final characterization using electron capture dissociation (ECD) combined with high resolution and high mass accuracy experiments on an FT-ICR mass spectrometer is under progress. [6]


Supported by Ministry of Education (MSM 113100001), by the Institutional Research Concept AVOZ5020903, and by Grant Agency of Czech Republic (No. 203/04/1045).


[1] V.M. Hearn, G.M. Escott, E. Glyn, V. Evans, D.J.Adams, Microbios, 93 (1998) 85-104.

[2] L. Weignerová, P. Vavrušková, A. Pišvejcová, J. Thiem, V. Křen, Carbohydr.Res., 338 (2003) 1003-1008. [3] L. Hušáková, E. Herkommerová-Rajnochová, T.Semeňuk, M. Kuzma, J. Rauvolfová, V. Přikrylová, R. Ettrich, O. Plíhal, K. Bezouška, V. Křen, Adv.Synth.Catal., 345 (2003) 735-742. [4] O. Plíhal et al., manuscript in preparation. [5] J. Sklenář et al., manuscript in preparation. [6] P. Man et al., manuscript in preparation.