title: Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum.
abstract: 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 (alpha/beta)(8)-barrel fold enzymes to recognize beta-1,4-galactosylated and alpha-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, beta-1,4-galactosylated and alpha-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.
authors.affiliation: VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland. harry.boer@vtt.fi
authors.forename: Harry
authors.initials: H
authors.lastname: Boer
authors.forename: Nana
authors.initials: N
authors.lastname: Munck
authors.forename: Jari
authors.initials: J
authors.lastname: Natunen
authors.forename: Gerd
authors.initials: G
authors.lastname: Wohlfahrt
authors.forename: Hans
authors.initials: H
authors.lastname: Söderlund
authors.forename: Ossi
authors.initials: O
authors.lastname: Renkonen
authors.forename: Anu
authors.initials: A
authors.lastname: Koivula
mesh_terms: D000818:Animals; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D048271:Chitosan / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005643:Fucose / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism*; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D005643:Fucose; D048271:Chitosan; D002688:Chitinases