Abstract
The reaction mechanism of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) was studied by site-directed mutagenesis and x-ray crystallography. An inactive mutant of BiSP (E232Q) was co-crystallized with sucrose. The structure revealed a substrate-binding mode comparable with that seen in other related sucrose-acting enzymes. Wild-type BiSP was also crystallized in the presence of sucrose. In the dimeric structure, a covalent glucosyl intermediate was formed in one molecule of the BiSP dimer, and after hydrolysis of the glucosyl intermediate, a beta-D-glucose product complex was formed in the other molecule. Although the overall structure of the BiSP-glucosyl intermediate complex is similar to that of the BiSP(E232Q)-sucrose complex, the glucose complex discloses major differences in loop conformations. Two loops (residues 336-344 and 132-137) in the proximity of the active site move up to 16 and 4 A, respectively. On the basis of these findings, we have suggested a reaction cycle that takes into account the large movements in the active-site entrance loops.
| Original language | English |
|---|---|
| Journal | Journal of Biological Chemistry |
| Volume | 281 |
| Issue number | 46 |
| Pages (from-to) | 35576-84 |
| Number of pages | 9 |
| ISSN | 0021-9258 |
| DOIs | |
| Publication status | Published - 2006 |
Keywords
- Bifidobacterium
- Binding Sites
- Carbohydrate Conformation
- Glucosyltransferases
- Models, Molecular
- Protein Conformation
- Sucrose