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Bone adhesives offer distinct advantages over the use of screws to attached internal fixation plates (IFPs). As the chemical composition of bone is similar to dentine, it is possible that the types of monomers used to make dentine adhesives could be utilised to affix IFPs to bone. The ability to attach a bio-resorbable IFP to porcine bone was assessed for the monomer 10-methacryloyloxydecyl dihydrogen phosphate (MDP), used either as a homopolymer or a copolymer with urethane dimethacrylate (MDP + U). Additionally, the addition of a priming step (MDP + U + P) was evaluated. The chemical interactions of the monomers with bone were assessed using XRD and imaged using TEM, revealing the formation of nano-layered structures with the MDP primer, something we believe has not been reported on bone. In a 6-week artificial aging study both MDP + U and MDP + U + P demonstrated adequate shear bond strength to affix bio-resorbable IFPs. The cytotoxicity profiles of the adhesive formulations were determined using indirect and direct contact with MC3T3 cells, with indirect conditions suggesting the MDP + U + P is as cytocompatible as the resorbable IFP. The findings of this study suggest our newly developed adhesive has the potential to be used as a bone adhesive to affix bioresorbable IFPs. [ABSTRACT FROM AUTHOR]

Research has been conducted on the phosphotriesterase isolated from Pseudomonas diminuta. The high-resolution X-ray structures of the substituted phosphotriesterase forms crystallized in the substrate analogues absence are described.

Research has been conducted on the molecular structure of methylmalonyl CoA decarboxylase from Escherichia coli. The use of the X-ray crystallographic analysis is described.

The molecular structures of the bis(Mg2+)-adenosine triphosphate-oxalate complex of pyruvate kinase were examined. Pyruvate kinase from rabbit muscle was cocrystallized as a complex with MgIIATP, oxalate, magnesium ion and either potassium ion or sodium ion. Results showed that there were negligible differences in the structures of the complexes with potassium ion and sodium ion. It was also found that there is a variety of subunit conformations within the asymmetric unit.

The functional properties of four site-directed vegetative cell ferredoxin (Fd) mutants, C41S, C46S, C49S and C79S, and an X-ray crystallographic structure for C49S are presented. Each mutant has the ability to rapidly accept an electron from deazariboflavin semiquinone and to transfer an electron from its reduced form to oxidized ferredoxin:NADP+ reductase (FNR) although they are less reactive toward FNR and are less stable in solution than is wt Fd. Results suggest that the reactivity of the mutants is not correlated with altered reduction potentials.

Nonconservative mutations of the critical residues S47, F65, and E94, on the surface of ferredoxin have large parallel effects on the reduction potential and the electron-transfer reactivity of the (2Fe-2S) cluster. The reduction potential changes are not the principal factor governing electron transfer reactivity. The specific orientation of the proteins within the transient electron-transfer complex control the kinetic properties Anabaena Ferredoxin.

The three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3 is studied. Crystallographic analysis showed that the enzyme is a trimer and each subunit folds into two distinct motifs. The bigger, N-terminal domain has 10 strands of beta-pleated sheet that form two distinct layers which are almost perpendicular to each other. Alpha-helices are found on the sides of the layers of beta-sheet. The C-terminal domain is made of three amphiphilic alpha-helices. The two domains of the subunit are linked by a cation.

The phosphotriesterase from Pseudomonas diminuta requires a binuclear metal center for its activity. The enzyme contains two equivalents of zinc per subunit. Three-dimensional Zn2+/Zn2+ substituted structure of the enzyme indicates that the inhibitor diethyl 4-methylbenzylphosphonate binds to a site near the binuclear metal center. The activation of the nucleophilic water molecule which attacks the electrophilic phosphorus of the substrate occurs through interaction with the binuclear metal center.

An X-ray crystallographic analysis of the structure of kanamycin nucleotidyltransferase (KNTase) complexed with AMPCPP and kanamycin reveals that KNTase exists in a dimeric form. The enzyme exhibits both protein-antibiotic and protein-nucleotide interactions. The 4'-hydroxyl group of kanamycin which is appropriately aligned for a displacement attack at the phosphorus of the nucleotide is 5 angstrom apart. Glu at position 145 which is at the active site of the enzyme initially reacts with the kanamycin to activate the antibiotic for further attack at the phosphorus.

X-ray crystallographic studies of the cadmium substituted phosphotriesterase reveal the presence of ions, one ligated by two nitrogen and four oxygens forming a distorted octahedron and the other a trigonal bipyramid with two nitrogen- and three oxygen- containing ligands. Crystallization of the natural protein which has two zinc ions has not been possible. The enzyme has the rare capacity to hydrolyze common pesticides as well as the phosphofluoridates, though the exact mechanism is unknown, and it is an important weapon against chemical warfare.

Single crystal X-ray diffraction studies provide the three-dimensional structure at 2.1 angstroms resolution of phosphotriesterase, an organophosphate-degrading enzyme. The enzyme molecules contain an alpha/beta barrel with eight strands of parallel beta-pleated sheet and two antiparallel beta-strands at the C-terminus. This enzyme can mediate detoxification of organophosphate nerve agents.

Molecular replacement procedures were used to determine the three-dimensional structure of the high-potential iron-sulfur protein (iso-form II), isolated from Ectothiorhodospira vacuolata. The molecules revealed reverse turns that enveloped the iron-sulfur cluster with the 34, 37, 51 and 65 cysteine residues, binding the metal center to the polypeptide chain. Fifty-seven solvent molecules were observed on the protein surface.

Molecular composition of kanamycin nucleotidyltransferase, initially separated from Staphylococcus aureus, was examined using X-ray crystallographic analysis to a resolution 3.0 angstroms. The mixed beta-pleated sheet composed of five strands was the main feature of the N-terminal area, while five alpha-helices were present in the C-terminal region. An ellipsoid with a deep cleft was produced when the two subunits combine together.

The crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor (VDR) in ternary complexes with a synthetic LXXLL-containing peptide and four ligands are determined. The differences in the biological activity of the 2-carbon-modified analogues are not reflected in the three-dimensional structure of the LBD of the VDR.

This article describes a proof of concept study designed to evaluate the potential of an in vivo three-dimensional printing route to support minimally invasive repair of the musculoskeletal system. The study uses a photocurable material to additively manufacture in situ a model implant and demonstrates that this can be achieved effectively within a clinically relevant timescale. The approach has the potential to be applied with a wide range of light-curable materials and with development could be applied to create functionally gradient structures in vivo. [ABSTRACT FROM AUTHOR]

The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N10-formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido- d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a β-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate. [ABSTRACT FROM AUTHOR]

Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (Npro) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, Npro is no longer active as a protease. The released Npro suppresses the induction of the host's type-I interferon-α/β (IFN-α/β) response. Npro binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus Npro. Npro is structurally distinct from other known cysteine proteases and has a novel “clam shell” fold consisting of a protease domain and a zinc-binding domain. The unique fold of Npro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of Npro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of Npro's interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of Npro, and its role in subversion of host immune response. [ABSTRACT FROM AUTHOR]

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and 32P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group [ABSTRACT FROM AUTHOR]

Examines the molecular structure of methylmalonyl co-enzyme A (CoA) decarboxylase (MMCD) in the Escherichia coli. Evidence for an indirect involvement of MMCD in catalysis; Identification of MMCD active site region within one subunit of the hexameric region; Comparison and contrast with 4-chlorobenzoyl CoA dehalogenase.

Abstract: The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N2 during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10–15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0Å resolution using sulfur anomalous scattering. [Copyright &y& Elsevier]

Abstract: T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95Å resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1–RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein–protein interactions mediated by the RNP motifs of TIA-1–RRM2. Importantly, the TIA-1–RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding. [Copyright &y& Elsevier]

Abstract: We have successfully prepared E- and Z- isomers of 17–20 dehydro analogs of 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD). Both isomers bind to the recombinant rat vitamin D receptor (VDR) with high affinity. The Z-isomer (Vit-III 17–20Z) displays activity in vivo and in vitro that is similar to 2MD. The in vitro activity of the E-isomer (Vit-III 17–20E) is comparable to the natural hormone, though in vivo this analog is significantly less calcemic. Crystal structures of the rat VDR ligand binding domain complexed with the analogs demonstrate that the Vit-III 17–20Z analog is oriented almost identically to 2MD, with only minor differences induced by the planar configuration around the C17–C20 double bond. The Vit-III 17–20E analog is oriented in a conformation distinct from both 2MD and the natural hormone. The structural comparisons suggest that the position of C21 in the ligand binding site may be an important determinant of biological activity. [Copyright &y& Elsevier]