Importance of Trp59 and Trp60 in chitin-binding, hydrolytic, and antifungal activities of Streptomyces griseus chitinase C.
The chitin-binding domain of Streptomyces griseus chitinase C (ChBD(ChiC)) belongs to CBM family 5. Only two exposed aromatic residues, W59 and W60, were observed in ChBD(ChiC), in contrast to three such residues on CBD(Cel5) in the same CBM family. To study importance of these residues in binding activity and other functions of ChBD(ChiC), site-directed mutagenesis was carried out. Single (W59A and W60A) and double (W59A/W60A) mutations abolished the binding activity of ChiC to colloidal chitin and decreased the hydrolytic activity toward not only colloidal chitin but also a soluble high Mr substrate, glycol chitin. Interaction of ChBD(ChiC) with oligosaccharide was eliminated by these mutations. The hydrolytic activity toward oligosaccharide was increased by deletion of ChBD but not affected by these mutations, indicating that ChBD interferes with oligosaccharide hydrolysis but not through its binding activity. The antifungal activity was drastically decreased by all mutations and significant difference was observed between single and double mutants. Taken together with the structural information, these results suggest that ChBD(ChiC) binds to chitin via a mechanism significantly different from CBD(Cel5), where two aromatic residues play major role, and contributes to various functions of ChiC. Sequence comparison indicated that ChBD(ChiC)-type CBMs are dominant in CBM family 5.
Itoh
Yoshikane
Y
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan.
Watanabe
Jun
J
Fukada
Harumi
H
Mizuno
Ryoji
R
Kezuka
Yuichiro
Y
Nonaka
Takamasa
T
Watanabe
Takeshi
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D010940:Plant Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013305:Streptomyces griseus / Q000201:enzymology*; D014242:Trichoderma / Q000187:drug effects / Q000254:growth & development; D014364:Tryptophan / Q000235:genetics* / Q000502:physiology*
D000935:Antifungal Agents; D009844:Oligosaccharides; D010940:Plant Proteins; D002686:Chitin; D014364:Tryptophan; D002688:Chitinases; C119825:chitinase C