Glycobiology Advance Access originally published online on June 30, 2004
Glycobiology 2004 14(12):1303-1313; doi:10.1093/glycob/cwh121
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Glycobiology vol. 14 no. 12 © Oxford University Press 2004; all rights reserved.
Differential recognition of animal type ß4-galactosylated and 
3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum
4 VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland, and 5 University of Helsinki, Helsinki, Finland
Received on June 2, 2004; accepted on June 28, 2004
We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (
/ß)8-barrel fold enzymes to recognize ß-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 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 catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, ß-1,4-galactosylated and -1,3-fucosylated chito-oligosaccharides. The cleavage data give 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. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.
2 Present address: Glykos Finland ltd, Viikinkaari 4, 00790 Helsinki, Finland
3 Present address: Orion Pharma, P.O. Box 65, FIN-02101 Espoo, Finland
1 To whom correspondence should be addressed; e-mail: harry.boer{at}vtt.fi
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