Abstract
The domestic silkworm Bombyx mori expresses two sucrose-hydrolyzing 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 α-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 Å resolutions. These structures revealed that the conformations of amino acid residues around subsite -1 are notably different at each step of the hydrolytic reaction. Such changes have not been previously reported among GH13 enzymes, including exo- and endo-acting hydrolases, such as α-glucosidases and α-amylases. Amino acid residues at subsite +1 are not conserved in BmSUH and other GH13_17 α-glucosidases, but subsite -1 residues are absolutely conserved. Substitutions in three subsite +1 residues, Gln191, Tyr251, and Glu440, 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 α-glucosidases.
Highlights
Sucrose, a-D-glucopyranosyl-(1!2)-b-D-fructofuranoside, is ubiquitously distributed in plants and is utilized as a carbon source by many organisms
The N-terminally His-tagged B. mori sucrose-specific hydrolases (SUHs) (BmSUH) without its transmembrane region was expressed in E. coli BL21 (DE3)
When host cells were cultured and induced in LB supplemented with 10 mM CaCl2, the final yield of recombinant BmSUH reached ;3 mg from 1 liter of the culture, suggesting that BmSUH may require calcium ion for proper folding
Summary
A-D-glucopyranosyl-(1!2)-b-D-fructofuranoside, is ubiquitously distributed in plants and is utilized as a carbon source by many organisms. We examined the crystal structures of B. mori SUH (BmSUH) in an apo form and in complexes with ligands, including substrates and inhibitors. Structure of GH13_17 sucrose hydrolase covalent intermediate was determined using a synthetic substrate, revealing conformational changes of the enzyme and the complete conformational itinerary of substrates during hydrolysis.
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