A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide.
YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.
Itoh
Takafumi
T
Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Ochiai
Akihito
A
Mikami
Bunzo
B
Hashimoto
Wataru
W
Murata
Kousaku
K
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000145:classification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013379:Substrate Specificity
D004187:Disaccharides; D006026:Glycoside Hydrolases; D000117:Acetylglucosamine