Structural Rearrangements of Sucrose Phosphorylase from Bifidobacterium adolescentis during Sucrose Conversion

O. Mirza, L.K. Skov, D. Sprogoe, L.A.M. van den Broek, G. Beldman, J.S. Kastrup, M. Gajhede

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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 -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 4Å, 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 languageEnglish
Pages (from-to)35576-35584
JournalJournal of Biological Chemistry
Volume281
Issue number46
DOIs
Publication statusPublished - 2006

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sucrose phosphorylase
Sucrose
Catalytic Domain
Glucose
Mutagenesis
Crystallography
Molecules
Site-Directed Mutagenesis
Dimers
Conformations
Hydrolysis
X-Rays
X rays
Bifidobacterium adolescentis
Substrates
Enzymes

Keywords

  • alpha-amylase family
  • crystal-structure
  • neisseria-polysaccharea
  • amylosucrase
  • mechanism
  • glucose
  • complex
  • enzyme

Cite this

Mirza, O. ; Skov, L.K. ; Sprogoe, D. ; van den Broek, L.A.M. ; Beldman, G. ; Kastrup, J.S. ; Gajhede, M. / Structural Rearrangements of Sucrose Phosphorylase from Bifidobacterium adolescentis during Sucrose Conversion. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 46. pp. 35576-35584.
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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 -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{\AA}, 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.",
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Structural Rearrangements of Sucrose Phosphorylase from Bifidobacterium adolescentis during Sucrose Conversion. / Mirza, O.; Skov, L.K.; Sprogoe, D.; van den Broek, L.A.M.; Beldman, G.; Kastrup, J.S.; Gajhede, M.

In: Journal of Biological Chemistry, Vol. 281, No. 46, 2006, p. 35576-35584.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Structural Rearrangements of Sucrose Phosphorylase from Bifidobacterium adolescentis during Sucrose Conversion

AU - Mirza, O.

AU - Skov, L.K.

AU - Sprogoe, D.

AU - van den Broek, L.A.M.

AU - Beldman, G.

AU - Kastrup, J.S.

AU - Gajhede, M.

PY - 2006

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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 -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 4Å, 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 -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 4Å, 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 - alpha-amylase family

KW - crystal-structure

KW - neisseria-polysaccharea

KW - amylosucrase

KW - mechanism

KW - glucose

KW - complex

KW - enzyme

U2 - 10.1074/jbc.M605611200

DO - 10.1074/jbc.M605611200

M3 - Article

VL - 281

SP - 35576

EP - 35584

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 46

ER -