Structure–function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 exo-α-glucosidases
Takatsugu Miyazaki, Enoch Y. Park
Abstract
The domestic silkworm Bombyx mori expresses two sucrosehydrolyzing enzymes, BmSUH and BmSUC1, belonging to glycoside hydrolase family 13 subfamily 17 (GH13_17) and GH32, respectively. BmSUH has little activity on maltooligosaccharides, whereas other insect GH13_17 a-glucosidases are active on sucrose and maltooligosaccharides. Little is currently known about the structural mechanisms and substrate specificity of GH13_17 enzymes. In this study, we examined the crystal structures of BmSUH without ligands; in complexes with substrates, products, and inhibitors; and complexed with its covalent intermediate at 1.60-1.85 resolutions. These structures revealed that the conformations of amino acid residues around subsite 21 are notably different at each step of the hydrolytic reaction. Such changes have not been previously reported among GH13 enzymes, including exoand endo-acting hydrolases, such as a-glucosidases and a-amylases. Amino acid residues at subsite 11 are not conserved in BmSUH and other GH13_17 a-glucosidases, but subsite 21 residues are absolutely conserved. Substitutions in three subsite 11 residues, Gln 191 , Tyr 251 , and Glu 440 , decreased sucrose hydrolysis and increased maltase activity of BmSUH, indicating that these residues are key for determining its substrate specificity. These results provide detailed insights into structure-function relationships in GH13 enzymes and into the molecular evolution of insect GH13_17 a-glucosidases.