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 ␤-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 Å , 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.

Amino Acids in Conserved Region II Are Crucial to Substrate Specificity, Reaction Velocity, and Regioselectivity in the Transglucosylation of Honeybee GH-13 α-Glucosidases

“…The sequence motif DAxxY/F (corresponding to Asp223-Tyr227 of HBG-III) frequently occurs in GH-13 enzymes belonging to subfamilies 4 (sucrose hydrolases), 16 (trehalose synthases), 17 (-glucosidases), 18 (sucrose phosphorylases), and 23 (-glucosidases). The protein structures of Tyr-harboring enzymes, sucrose hydrolase derived from Xanthomonas campestris 27) and sucrose phosphorylase from Bifidobacterium adolescentis, 28) indicate that the side chain of Tyr has no direct contact with a fructose moiety of sucrose. It is accommodated on the far side of the pocket from the equivalent residues, 27) as observed for the isomaltotriose-binding mode of dextran glucosidase.…”

“…1) is often found in subfamilies 1,2,5,15,17,19,24,27,28,31,32, and 36, which include -1,4-linkage-specific enzymes such as -amylases, cyclomaltodextrin glucanotransferases, and -glucosidases. Most -amylases and cyclomaltodextrin glucanotransferases have dipeptide Lys-His at the C-terminal end of the motif.…”

“…Pro226-Tyr227 of HBG-III and their equivalents are indicated by bold-faced letters. The partial sequences, listed from the top, are of -glucosidase isozymes III, II, and I from Apis mellifera (HBG-III, HBG-II, and HBG-I respectively), 6,12) Taka-amylase from Aspergillus oryzae, 25) Bacillus subtilisamylase, 26) sucrose hydrolase SUH from Xanthomonas axonopodis, 27) sucrose phosphorylase from Bifidobacterium adolescentis, 28) amylosucrase from Neisseria polysaccharea, 30) trehalulose synthase (MutB) from Psuedomonas mesoacidophila, 31) and dextran glucosidase (SmDG) from Streptococcus mutans. 29) hydrolysate (6 N HCl at 110 C for 24 h) by the ninhydrin colorimetric method using an amino acid analyzer (AminoTac JLC/500V, JEOL, Tokyo).…”

Sucrose phosphorylases catalyze transglycosylation reactions on carboxylic acid compounds

“…These results support the hypothesis that the undissociated OH form of benzoic acid can be used as an effective acceptor molecule by the enzymes. This is quite reasonable from the view point of the proposed catalytic mechanism of phosphorolysis by sucrose phosphorylase from Bifidobacterium adolescentis that protonated phosphate group is necessary for binding to the catalytic domain of the enzyme (Mirza et al 2006). The transfer efficiency of the transglycosylation reaction of sucrose phosphorylase from L. mesenteroides was much lower than that of sucrose phosphorylase from S. mutans at pH 3.9.…”

Novel Transglucosylating Reaction of Sucrose Phosphorylase to Carboxylic Compounds

“…This is quite reasonable from the viewpoint of the proposed catalytic mechanism of the transglycosylation reaction toward phosphate by sucrose phosphorylase from Bifidobacterium adolescentis. 25) It suggested that sucrose phosphorylase can react with HPO4 2 or H2PO4 but not with PO4 3 because a protonated phosphate group is necessary for its binding to the catalytic domain of the enzyme. We considered that the glucosyltransfer reaction of sucrose phosphorylase was very slow around neutral pH, since the reaction mixture contained only a small amount of the available form of acetic acid.…”