Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium.
The basidiomycete Phanerochaete chrysosporium produces xyloglucanase Xgh74B, which has the glycoside hydrolase (GH) family 74 catalytic domain and family 1 carbohydrate-binding module, in cellulose-grown culture. The recombinant enzyme, which was heterologously expressed in the yeast Pichia pastoris, had high hydrolytic activity toward xyloglucan from tamarind seed (TXG), whereas other beta-1,4-glucans examined were poor substrates for the enzyme. The existence of the carbohydrate-binding module significantly affects adsorption of the enzyme on crystalline cellulose, but has no effect on the hydrolysis of xyloglucan, indicating that the domain may contribute to the localization of the enzyme. HPLC and MALDI-TOF MS analyses of the hydrolytic products of TXG clearly indicated that Xgh74B hydrolyzes the glycosidic bonds of unbranched glucose residues, like other GH family 74 xyloglucanases. However, viscometric analysis suggested that Xgh74B hydrolyzes TXG in a different manner from other known GH family 74 xyloglucanases. Gel permeation chromatography showed that Xgh74B initially produced oligosaccharides of degree of polymerization (DP) 16-18, and these oligosaccharides were then slowly hydrolyzed to final products of DP 7-9. In addition, the ratio of oligosaccharides of DP 7-9 versus those of DP 16-18 was dependent upon the pH of the reaction mixture, indicating that the affinity of Xgh74B for the oligosaccharides of DP 16-18 is affected by the ionic environment at the active site.
Ishida
Takuya
T
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
Yaoi
Katsuro
K
Hiyoshi
Ayako
A
Igarashi
Kiyohiko
K
Samejima
Masahiro
M
D001665:Binding Sites; D002240:Carbohydrate Sequence; D018076:DNA, Complementary / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000201:enzymology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D005656:Fungal Proteins; D005936:Glucans; D011994:Recombinant Proteins; D014990:Xylans; C029353:xyloglucan; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase