Identification and Characterization of γ-Glutamylamine Cyclotransferase,an Enzyme Responsible for γ-Glutamyl-?-lysine Catabolism

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu⁸²). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.     

Plastid division: across time and space

Plastid division is executed by the coordinated action of at least two molecular machineries--an internal machinery situated on the stromal side of the inner envelope membrane that was contributed by the cyanobacterial endosymbiont from which plastids evolved, and an external machinery situated on the cytosolic side of the outer envelope membrane that was contributed by the host. Here we review progress in defining the components of the plastid division complex and understanding the mechanisms of envelope constriction and division-site placement in plants. We also highlight recent work identifying the first molecular linkage between the internal and external division machineries, shedding light on how their mid-plastid positioning is coordinated across the envelope membranes. Little is known about the mechanisms that regulate plastid division in plant cells, but recent studies have begun to hint at potential mechanisms.     

Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic β-hematin (βH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to βH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.     

A multiple in silico program approach for the prediction of mutagenicity from chemical structure

We have conducted an evaluation of three of the most widely used commercial toxicity prediction programs, Toxicity Prediction by Komputer Assisted Technology (TOPKAT), Deductive Estimation of Risk from Existing Knowledge (DEREK) for Windows (DfW) and CASETOX. The three programs were evaluated for their ability to predict Ames test mutagenicity using 520 proprietary drug candidate (Test set 1) and 94 commercial (Test set 2) compounds. The study demonstrates that these three commercially available programs are useful, with limitations in their ability to predict mutagenicity over a wide range of chemical space, i.e. global predictivity. Individually, each of the programs performed at an acceptable level for overall accuracy, i.e. the ability to predict the correct outcome. However, analysis of the predictions indicates that the overall accuracy figure is heavily weighted by the ability of the programs to correctly predict non-mutagens, whereas none of the programs individually performed well in the prediction of novel mutagenic structures, i.e. Ames positive compounds. The performance of these programs' in predicting Ames positive mutagens appeared to be independent of the chemical utility of the compound, i.e. industrial, agricultural or pharmaceutical. The combination of program predictions provided some improvement in overall accuracy, sensitivity and specificity.     

Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain

A complex of the three (αεθ) core subunits and the β₂ sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β₂ in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β₂ complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β₂ demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β₂ replicase complex with primer-template DNA combine all available structural data.     

Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic

Of the 200+ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a non-endemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in non-O1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.     

A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent biological effects against multiple myeloma cells

A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC₅₀’s <5 μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold’s history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.     

The use of preserved tissue in finite element modelling of fresh ovine vertebral behaviour

The aim of this study was to investigate whether the predicted finite element (FE) stiffness of vertebral bone is altered when using images of preserved rather than fresh tissue to generate specimen-specific FE models. Fresh ovine vertebrae were used to represent embalmed (n = 3) and macerated dry-bone (n = 3) specimens and treated accordingly. Specimens were scanned pre- and post-treatment using micro-computed tomography. A constant threshold level derived from these images was used to calculate the respective bone volume fraction (BV/TV) from which the conversion factor validated for fresh tissue was used to determine material properties that were assigned to corresponding FE models. Results showed a definite change in the BV/TV between the fresh and the preserved bone. However, the changes in the predicted FE stiffness were not generally greater than the variations expected from assignment of loading and boundary conditions. In conclusion, images of preserved tissue can be used to generate FE models that are representative of fresh tissue with a tolerable level of error.