The exo-1,3-beta-glucanase (Exg) from Candida albicans is involved in cell wall beta-D-glucan metabolism and morphogenesis through its hydrolase and transglycosidase activities. Previous work has shown that both these activities strongly favor beta-1,3-linkages. The E292S Exg variant displayed modest glycosynthase activity using alpha-D-glucopyranosyl fluoride (alpha-GlcF) as the donor and pNP-beta-D-glucopyranoside (pNPGlc) as the acceptor but surprisingly showed a marked preference for synthesizing beta-1,6-linked over beta-1,3- and beta-1,4-linked disaccharide products. With pNPXyl as the acceptor, the preference became beta-1,4 over beta-1,3. The crystal structure of the glycosynthase bound to both of its substrates, alpha-GlcF and pNPGlc, is the first such ternary complex structure to be determined. The results revealed that the donor bound in the -1 subsite, as expected, while the acceptor was oriented in the +1 subsite to facilitate beta-1,6-linkage, thereby supporting the results from solution studies. A second crystal structure containing the major product of glycosynthesis, pNP-gentiobiose, showed that the -1 subsite allows another docking position for the terminal sugar; i.e., one position is set up for catalysis, whereas the other is an intermediate stage prior to the displacement of water from the active site by the incoming sugar hydroxyls. The +1 subsite, an aromatic "clamp", permits several different sugar positions and orientations, including a 180 degrees flip that explains the observed variable regiospecificity. The p-nitrophenyl group on the acceptor most likely influences the unexpectedly observed beta-1,6-specificity through its interaction with F229. These results demonstrate that tailoring the specificity of a particular glycosynthase depends not only on the chemical structure of the acceptor but also on understanding the structural basis of the promiscuity of the native enzyme.

Page s 135glycosyl hydrolase with exo-1,3-β-glucanase activity was isolated and its gene subsequently over-expressed in E.coli.The sequence showed it to be a GH3 family member, closely related to barley exo-1,3-1,4-β-glucanase [1] in two of its three domains.This was confirmed by crystallographic analysis which also showed how the additional C-terminal domain interacts with the N-terminal domain.Activity studies demonstrated the extra domain was essential for enzyme activity.

The exo-ß-1,3-glucanase from Candida albicans (Exg) functions in cell wall remodelling through its glucosyl hydrolase and transferase activities.As with other family 5 glycosyl hydrolases Exg possesses catalytic glutamate residues acting as nucleophile (E292) and proton donor (E192).The entrance to the active site pocket is flanked by a pair of antiparallel phenylalanine residues (F144 and F258).Crystallographic analysis of enzyme:inhibitor complexes has identified protein-sugar interactions at the -1 site and, to a lesser extent, at the +1/+2 aromatic entranceway [1].We have made various mutants to try and better identify sugar-binding sites, to alter the ratio of hydrolysis to transfer and to compare substrate specificity for hydrolase and glucosynthase reactions.Mutations at the Phe-Phe gateway demonstrated that aromaticity must be preserved and that the transferase reaction was more sensitive to mutation, consistent with two docking events.The catalytically disabled mutant E292Q did not show coherent electron density to indicate stable binding of oligosaccharide substrates but unexpectedly revealed an external glucose binding site that may provide a foothold for the yeast cell wall Dglucan.Exg was converted to a glucosynthase via E292S and crystallized in the presence of donor 1-fluoro-α-Dglucose and acceptor p-nitrophenyl-ß-D-glucopyranoside [2].The two sugars were oriented such that donor sugar C1 was close to acceptor O6, consistent with nmr analysis of solution products which showed the main product to be ß-1,6-linked disaccharide and not ß-1,3-as expected.

BMPRII is a type II TGF-β serine threonine kinase receptor which is integral to the bone morphogenetic protein (BMP) signalling pathway. It is known to bind BMP and growth differentiation factor (GDF) ligands, and has overlapping ligand specificity with the activin type II receptor, ActRII. In contrast to activin and TGF-β type ligands, BMPs bind to type II receptors with lower affinity than type I receptors. Crystals of the BMPRII ectodomain were grown in two different forms, both of which diffracted to high resolution. The tetragonal form exhibited some disorder, whereas the entire polypeptide was seen in the orthorhombic form. The two structures retain the basic three-finger toxin fold of other TGF-β receptor ectodomains, and share the main hydrophobic patch used by ActRII to bind various ligands. However, they present different conformations of the A-loop at the periphery of the proposed ligand-binding interface, in conjunction with rearrangement of a disulfide bridge within the loop. This particular disulfide (Cys94–Cys117) is only present in BMPRII and activin receptors, suggesting that it is important for their likely shared mode of binding. Evidence is presented that the two crystal forms represent ligand-bound and free conformations of BMPRII. Comparison with the solved structure of ActRII bound to BMP2 suggests that His87, unique amongst TGF-β receptors, may play a key role in ligand recognition.

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.