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