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 (α/β)8-barrel fold enzymes to recognize β-1,4-galactosylated and α-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, β-1,4-galactosylated and α-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.
- (alfa/beta)8-barrel fold
- molecular modeling
- substrate specificity
Boer, H., Munck, N., Natunen, J., Wohlfahrt, G., Söderlund, H., Renkonen, O., & Koivula, A. (2004). Differential recognition of animal type β4-galactosylated and α3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum. Glycobiology, 14(12), 1303 - 1313. https://doi.org/10.1093/glycob/cwh121