Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis.

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d-Fructose and a-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 Å resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate-dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B‘, and C. Domain A comprises the (ß/a)8-barrel common to family 13. The catalytic active-site residues (Asp192 and Glu232) are located at the tips of ß-sheets 4 and 5 in the (ß/a)8-barrel, as required for family 13 members. The topology of the B‘ domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 Å2 per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active sites

Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d Fructose and amp; 945; d glucose 1 phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis BiSP refined at 1.77 resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B , and C. Domain A comprises the amp; 946; amp; 945; 8 barrel common to family 13. The catalytic active site residues Asp192 and Glu232 are located at the tips of amp; 946; sheets 4 and 5 in the amp; 946; amp; 945; 8 barrel, as required for family 13 members. The topology of the B domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 2 per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active site

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 -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

Ionotropic Glutamate Receptor GluA2 in Complex with Bicyclic Pyrimidinedione Based Compounds When Small Compound Modifications Have Distinct Effects on Binding Interactions

S 2 Amino 3 5 methyl 3 hydroxyisoxazol 4 yl propanoic acid AMPA receptors comprise an important class of ionotropic glutamate receptors activated by glutamate in the central nervous system. These receptors have been shown to be involved in brain diseases, for example, Alzheimer s disease and epilepsy. To understand the functional role of AMPA receptors at the molecular level and their potential as targets for drugs, development of tool compounds is essential. We have previously reported the synthesis of six bicyclic pyrimidinedione based analogues of willardiine with differences limited to the pyrimidinedione fused five membered rings. Despite minor molecular differences, we observed gt;500 fold difference in binding affinity of the compounds at full length GluA2. Here, we report binding affinities and the binding mode of these compounds at the ligand binding domain of GluA2 using X ray crystallography. The structures revealed similar binding modes, with distinct differences in the interaction between GluA2 and the compounds. The methylene 2 and sulfur 3 containing compounds showed the greatest binding affinities. Changing the dihydrothiophene 3 into pyrrolidine 4 , N methyl pyrrolidine 5 , or dihydrofuran 6 induced flexibility in the position of a binding site water molecule and changes in the hydrogen bonding network between compound, water, and GluA2. This might be essential for explaining the reduced binding affinity of these compounds. The weakest binding affinity was observed when the aliphatic oxygen containing dihydrofuran 6 was changed into an aromatic furan system 7 . Molecular docking studies revealed two possible orientations of 7, whereas only one binding mode was observed for the other analogues. This could likely contribute to the weakest binding affinity of 7 at GluA

Sucrose phosphorylase as cross-linked enzyme aggregate: improved thermal stability for industrial applications

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60 degrees C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17 degrees C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60 degrees C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity