Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum.
Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites -4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 microM whereas no binding of chitinbiose, -triose or 3'-sialyl-N-acetyllactosamine (Neu5Acalpha-3Galbeta-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (K(d) = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites -2 and -3 and particularly the stacking interactions by tryptophanes at subsites -3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNbeta-4(GlcNAc)(4) by providing a counter charge through substitution of residue T133 at subsite -3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galbeta-4(GlcNAc)(4).
Lienemann
Michael
M
VTT Technical Research Centre of Finland, FI-02044 VTT, Finland.
Boer
Harry
H
Paananen
Arja
A
Cottaz
Sylvain
S
Koivula
Anu
A
D001665:Binding Sites; D055162:Biocatalysis; D002240:Carbohydrate Sequence; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D014242:Trichoderma / Q000201:enzymology*
D005656:Fungal Proteins; D009844:Oligosaccharides; D002688:Chitinases