Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion

TY - JOUR

T1 - Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion

AU - Mirza, Osman

AU - Henriksen, Lars Skov

AU - Sprogøe, Desiree

AU - van den Broek, Lambertus A M

AU - Beldman, Gerrit

AU - Kastrup, Jette S

AU - Gajhede, Michael

PY - 2006

Y1 - 2006

N2 - 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.

AB - 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.

KW - Bifidobacterium

KW - Binding Sites

KW - Carbohydrate Conformation

KW - Glucosyltransferases

KW - Models, Molecular

KW - Protein Conformation

KW - Sucrose

U2 - 10.1074/jbc.M605611200

DO - 10.1074/jbc.M605611200

M3 - Journal article

C2 - 16990265

SN - 0021-9258

VL - 281

SP - 35576

EP - 35584

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 46

ER -