Structure and function of the thermostable L-asparaginase from Thermococcus kodakarensis.

L-Asparaginases catalyse the hydrolysis of asparagine to aspartic acid and ammonia. In addition, L-asparaginase is involved in the biosynthesis of amino acids such as lysine, methionine and threonine. These enzymes have been used as chemotherapeutic agents for the treatment of acute lymphoblastic leukaemia and other haematopoietic malignancies since the tumour cells cannot synthesize sufficient L-asparagine and are thus killed by deprivation of this amino acid. L-Asparaginases are also used in the food industry and have potential in the development of biosensors, for example for asparagine levels in leukaemia. The thermostable type I L-asparaginase from Thermococcus kodakarensis (TkA) is composed of 328 amino acids and forms homodimers in solution, with the highest catalytic activity being observed at pH 9.5 and 85°C. It has a Km value of 5.5 mM for L-asparagine, with no glutaminase activity being observed. The crystal structure of TkA has been determined at 2.18 Å resolution, confirming the presence of two α/β domains connected by a short linker region. The N-terminal domain contains a highly flexible β-hairpin which adopts `open' and `closed' conformations in different subunits of the solved TkA structure. In previously solved L-asparaginase structures this β-hairpin was only visible when in the `closed' conformation, whilst it is characterized with good electron density in all of the subunits of the TkA structure. A phosphate anion resides at the active site, which is formed by residues from both of the neighbouring monomers in the dimer. The high thermostability of TkA is attributed to the high arginine and salt-bridge content when compared with related mesophilic enzymes

Structure of the 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis

Structure and function of the type III pullulan hydrolase from Thermococcus kodakarensis

Pullulan-hydrolysing enzymes, more commonly known as debranching enzymes for starch and other polysaccharides, are of great interest and have been widely used in the starch-saccharification industry. Type III pullulan hydrolase from Thermococcus kodakarensis (TK-PUL) possesses both pullulanase and α-amylase activities. Until now, only two enzymes in this class, which are capable of hydrolysing both α-1,4- and α-1,6-glycosidic bonds in pullulan to produce a mixture of maltose, panose and maltotriose, have been described. TK-PUL shows highest activity in the temperature range 95–100°C and has a pH optimum in the range 3.5–4.2. Its unique ability to hydrolyse maltotriose into maltose and glucose has not been reported for other homologous enzymes. The crystal structure of TK-PUL has been determined at a resolution of 2.8 Å and represents the first analysis of a type III pullulan hydrolyse. The structure reveals that the last part of the N-terminal domain and the C-terminal domain are significantly different from homologous structures. In addition, the loop regions at the active-site end of the central catalytic domain are quite different. The enzyme has a well defined calcium-binding site and possesses a rare vicinal disulfide bridge. The thermostability of TK-PUL and its homologues may be attributable to several factors, including the increased content of salt bridges, helical segments, Pro, Arg and Tyr residues and the decreased content of serine

Fatores associados a sintomas depressivos em estudantes do ensino médio de São Paulo, Brasil

OBJECTIVE: To assess factors associated with depression symptoms in high school students. METHODS: A cross-sectional study involving high school students was conducted in the city of São Paulo, Brazil, 2001. A total of 724 students aged 14-18 years answered questionnaires on life and health conditions. Another questionnaire was applied to working (44.8%) and unemployed (22.9%) students to collect information on working conditions. Factors associated to depressive disorders were analyzed using multiple logistic regression controlled for occupational status. RESULTS: Overall prevalence rate of depression was 7.5%. Rates according to gender were 39 (10.3%) in females and 15 (4.3%) in males. The multiple logistic regression analysis showed that factors associated with depressive disorders were: poor self-perception of health (OR=5.78), being female (OR = 2.45), and alcohol consumption (OR=2.35). CONCLUSIONS: The study results showed that sociodemographic, lifestyle and health variables were associated with symptoms of depression in this population. These ndings suggest that it is important to have mental health professionals available in high schools for early detection of mental conditions and student counseling.OBJETIVO: Investigar os fatores associados a sintomas depressivos em estudantes do ensino médio. MÉTODOS: Foi realizado estudo transversal com estudantes residentes no Município de São Paulo, Brasil, em 2001. O total de 724 estudantes com idades entre 14 e 18 anos preencheram questionários de condições de vida e saúde. Dentre eles, os estudantes trabalhadores (44,8%) e desempregados (22,9%) também responderam a um outro questionário de condições de trabalho. A regressão logística foi utilizada para determinar os fatores associados para apresentar distúrbios depressivos, utilizando-se a "situação ocupacional" para ajustar o modelo. RESULTADOS: A prevalência dos sintomas depressivos na população estudada foi de 7,5%; as taxas de acordo com o sexo foram de 39 (10,3%) e 15 (4,3%) nos adolescentes dos sexos feminino e masculino, respectivamente. A regressão logística mostrou que os fatores associados aos distúrbios depressivos são: baixo escore na auto-avaliação da saúde (OR=5,78), ser do sexo feminino (OR=2,45) e consumo de bebidas alcoólicas (OR=2,35). CONCLUSÕES: Os resultados mostraram que variáveis sociodemográficas, de estilo de vida e de saúde estavam associadas aos distúrbios depressivos. Esses achados sugerem a importância de que profissionais de saúde mental em escolas de ensino médio efetuem rastreio para reconhecer precocemente problemas mentais e fornecer aconselhamento aos estudantes.FAPESPCNP

The 1.1 angstrom resolution structure of a periplasmic phosphate-binding protein from Stenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement using MOLREP in which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in the MOLREP peak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. A BLAST search then indicated that the molecule was most probably a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of the S. maltophilia protein has been refined to an R factor of 10.15% and an Rfree of 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form

National observational study to evaluate the "cleanyourhands" campaign (NOSEC): a questionnaire based study of national implementation

INTRODUCTION: The number of national hand-hygiene campaigns has increased recently, following the World Health Organisation's (WHO) "Save Lives: clean your hands" initiative (2009), which offers hospitals a multi-component hand-hygiene intervention. The number of campaigns to be evaluated remains small. Most evaluations focus on consumption of alcohol hand rub (AHR). We are not aware of any evaluation reporting implementation of all campaign components. In a previously published report, we evaluated the effects of the English and Welsh cleanyourhands campaign (2004-8) on procurement of AHR and soap, and on selected healthcare associated infections. We now report on the implementation of each individual campaign component: provision of bedside AHR, ward posters, patient empowerment materials, audit and feedback, and guidance to secure institutional engagement. METHOD: SETTING: all 189 acute National Health Service (NHS) hospitals in England and Wales (December 2005-June 2008). Six postal questionnaires (five voluntary, one mandatory) were distributed to infection control teams six-monthly from 6 to 36 months post roll-out. Selection and attrition bias were measured. RESULTS: Response rates fell from 134 (71 %) at 6 months to 82 (44 %) at 30 months, rising to 167 (90 %) for the final mandatory one (36 months). There was no evidence of attrition or selection bias. Hospitals reported widespread early implementation of bedside AHR and posters and a gradual rise in audit. At 36 months, 90 % of respondents reported the campaign to be a top hospital priority, with implementation of AHR, posters and audit reported by 96 %, 97 % and 91 % respectively. Patient empowerment was less successful. CONCLUSIONS: The study suggests that all campaign components, apart from patient empowerment, were widely implemented and sustained. It supports previous work suggesting that adequate piloting, strong governmental support, refreshment of campaigns, and sufficient time to engage institutions help secure sustained implementation of a campaign's key components. The results should encourage countries wishing to launch coordinated national campaigns for hospitals to participate in the WHO's "Save Lives" initiative, which offers hospitals a similar multi-component intervention

What traits are carried on mobile genetic elements, and why?

Although similar to any other organism, prokaryotes can transfer genes vertically from mother cell to daughter cell, they can also exchange certain genes horizontally. Genes can move within and between genomes at fast rates because of mobile genetic elements (MGEs). Although mobile elements are fundamentally self-interested entities, and thus replicate for their own gain, they frequently carry genes beneficial for their hosts and/or the neighbours of their hosts. Many genes that are carried by mobile elements code for traits that are expressed outside of the cell. Such traits are involved in bacterial sociality, such as the production of public goods, which benefit a cell's neighbours, or the production of bacteriocins, which harm a cell's neighbours. In this study we review the patterns that are emerging in the types of genes carried by mobile elements, and discuss the evolutionary and ecological conditions under which mobile elements evolve to carry their peculiar mix of parasitic, beneficial and cooperative genes

Robust Detection of Hierarchical Communities from Escherichia coli Gene Expression Data

Determining the functional structure of biological networks is a central goal of systems biology. One approach is to analyze gene expression data to infer a network of gene interactions on the basis of their correlated responses to environmental and genetic perturbations. The inferred network can then be analyzed to identify functional communities. However, commonly used algorithms can yield unreliable results due to experimental noise, algorithmic stochasticity, and the influence of arbitrarily chosen parameter values. Furthermore, the results obtained typically provide only a simplistic view of the network partitioned into disjoint communities and provide no information of the relationship between communities. Here, we present methods to robustly detect coregulated and functionally enriched gene communities and demonstrate their application and validity for Escherichia coli gene expression data. Applying a recently developed community detection algorithm to the network of interactions identified with the context likelihood of relatedness (CLR) method, we show that a hierarchy of network communities can be identified. These communities significantly enrich for gene ontology (GO) terms, consistent with them representing biologically meaningful groups. Further, analysis of the most significantly enriched communities identified several candidate new regulatory interactions. The robustness of our methods is demonstrated by showing that a core set of functional communities is reliably found when artificial noise, modeling experimental noise, is added to the data. We find that noise mainly acts conservatively, increasing the relatedness required for a network link to be reliably assigned and decreasing the size of the core communities, rather than causing association of genes into new communities.Comment: Due to appear in PLoS Computational Biology. Supplementary Figure S1 was not uploaded but is available by contacting the author. 27 pages, 5 figures, 15 supplementary file