Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum.
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.
Boer
Harry
H
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland. harry.boer@vtt.fi
Munck
Nana
N
Natunen
Jari
J
Wohlfahrt
Gerd
G
Söderlund
Hans
H
Renkonen
Ossi
O
Koivula
Anu
A
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*
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D005643:Fucose; D048271:Chitosan; D002688:Chitinases