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.

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 alpha-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 A 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 (beta/alpha)(8)-barrel common to family 13. The catalytic active-site residues (Asp192 and Glu232) are located at the tips of beta-sheets 4 and 5 in the (beta/alpha)(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 A(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 sites.

The glucosyltransferase amylosucrase is structurally quite similar to the hydrolase ␣-amylase. How this switch in functionality is achieved is an important and fundamental question. The inactive E328Q amylosucrase variant has been co-crystallized with maltoheptaose, and the structure was determined by x-ray crystallography to 2.2 Å resolution, revealing a maltoheptaose binding site in the B-domain somewhat distant from the active site. Additional soaking of these crystals with maltoheptaose resulted in replacement of Tris in the active site with maltoheptaose, allowing the mapping of the ؊1 to ؉5 binding subsites. Crystals of amylosucrase were soaked with sucrose at different concentrations. The structures at ϳ2.1 Å resolution revealed three new binding sites of different affinity. The highest affinity binding site is close to the active site but is not in the previously identified substrate access channel. Allosteric regulation seems necessary to facilitate access from this binding site. The structures show the pivotal role of the B-domain in the transferase reaction. Based on these observations, an extension of the hydrolase reaction mechanism valid for this enzyme can be proposed. In this mechanism, the glycogen-like polymer is bound in the widest access channel to the active site. The polymer binding introduces structural changes that allow sucrose to migrate from its binding site into the active site and displace the polymer.

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Several enzymes acting on sucrose are found in glycoside hydrolase family 13 (the a Á/amylase family). They all transfer a glucosyl moiety from sucrose to an acceptor, but the products can be very different. The structure of a variant of one of these, the Glu328Gln mutant of Neisseria polysaccharea amylosucrase, has been determined in a ternary complex with sucrose and an oligosaccharide to 2.16 Å resolution using x-ray crystallography. Sucrose selectively binds in the active site and the oligosaccharide only binds at surface sites. When this structure is compared to structures of other enzymes acting on sucrose from glycoside hydrolase family 13, it is found that the active site residues are very similar around the glucose part of sucrose while much variation is seen around the fructose moiety.

(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 >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 GluA2.

Science Kitaku Kita-10jo, Nishi-4chome SAPPORO HOKKAIDO 060-0810 JAPAN Rod-shaped bacteria have three potential division sites in a cell. One of them is at mid-cell position, while the others are adjacent to the cell poles. Thus the precise placement of the FtsZ ring at the cell center is prerequisite for the accurate cell division. In Escherichia coli, the cell division site is determined by MinC, MinD, and MinE. MinD is a membrane-associated ATPase and is a septum site-determining factor through the activation and regulation of MinC and MinE. MinD is also known to undergo a rapid pole-to-pole oscillation movement in vivo. We have determined the three-dimensional structure of MinD from Pyrococcus horikoshii at 2.3Å resolution using the Se-Met MAD method. The crystal structure consists of a β -sheet with seven parallel and one antiparallel strands and eleven peripheral α -helices. Although we made no attempt to add ATP or ADP molecules in the purification or crystallization step, the electron density clearly shows that MinD contains bound ADP and magnesium-ion at the pocket close to the edge of the b-sheet on the surface of the MinD molecule. Structure analysis shows that MinD is most similar to nitrogenase iron protein, which is a member of the family of the P-loop containing nucleotide triphosphate hydrolase superfamily of proteins. Moreover MinD has a limited structural similarity with family of motor proteins. Although the tertiary structure of ATPase activity site is similar in these proteins, the overall topology is different. This fact suggests that MinD may work as a molecular switch in bacterial cell division. Structural and functional analysis of wild type and site-directed mutants of duck δ 1 and δ 2 crystallin are being used to investigate the enzymatic mechanism of argininosuccinate lyase (ASL). ASL catalyses the reversible breakdown of argininosuccinate to arginine and fumarate, a reaction involved in arginine biosynthesis and in the urea cycle. During evolution, overexpression of ASL in the avian eye lens, followed by gene duplication has resulted in two delta crystallins: δ 2, the ASL orthologue, and the enzymatically inactive delta1. The crystal structures of duck δ 1 and δ 2 crystallin have been solved. Structural comparisons indicate that both intra-and inter-species conformational changes occur in two regions of the N-terminal domain. As the residues implicated in the catalytic mechanism of δ 2/ASL are conserved in δ 1, we postulate that amino-acid substitutions in these two regions of δ 1 are important for substrate binding and hence catalysis. The crystal structure of δ 1 crystallin revealed the presence of a sulfate anion in the active site region that may mimic the fumarate moiety of the argininosuccinate substrate. This induced a large conformational change in the 280's loop and a rigid body motion in domain 3. The results suggest that Ser 281 may play the role of the acid catalyst in the enzymatic mechanism of δ 2/ASL. The crystal structure of the inactive S281A δ 2 mutant with arginino...

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