Glycobiology Advance Access published online on June 30, 2004
Glycobiology, doi:10.1093/glycob/cwh121
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1 VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland
* To whom correspondence should be addressed. E-mail: harry.boer{at}vtt.fi.
We report here the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum, and characterisation using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (
Accepted June 28, 2004
ORIGINAL ARTICLES
Differential recognition of animal type
4-galactosylated and 
3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum
2 University of Helsinki, Helsinki, Finland
Abstract 
/
)8-barrel fold enzymes to recognize -1,4-galactosylated and -1,3-fucosylated oligosaccharides, which are animal type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built in order to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyse some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, -1,4-galactosylated and -1,3-fucosylated, chito-oligosaccharides. The cleavage data gives experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove which provides more tight binding of the substrate at subsites (-2-1-+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites -4 and -3 of Chit42, and at the subsite -4 of Chit33. Fucose units can be accepted as a branch at the putative -3 and -4 sites of Chit33 and as a branch point at -3 of Chit42. This data provides a good starting point for our future protein engineering work aiming at chitinases with altered substrate-binding specificity./)8-barrel fold.
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