title: Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion.
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.
authors.affiliation: Biostructural Research Unit, Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Osman
authors.initials: O
authors.lastname: Mirza
authors.forename: Lars K
authors.initials: LK
authors.lastname: Skov
authors.forename: Desiree
authors.initials: D
authors.lastname: Sprogøe
authors.forename: Lambertus A M
authors.initials: LA
authors.lastname: van den Broek
authors.forename: Gerrit
authors.initials: G
authors.lastname: Beldman
authors.forename: Jette S
authors.initials: JS
authors.lastname: Kastrup
authors.forename: Michael
authors.initials: M
authors.lastname: Gajhede
mesh_terms: D001644:Bifidobacterium / Q000201:enzymology*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D005964:Glucosyltransferases / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013395:Sucrose / Q000378:metabolism*
chemical_list: D013395:Sucrose; D005964:Glucosyltransferases; C025901:sucrose phosphorylase