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 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

Structural studies of domain movement in active-site mutants of porphobilinogen deaminase from Bacillus megaterium.

The enzyme porphobilinogen deaminase (PBGD) is one of the key enzymes in tetrapyrrole biosynthesis. It catalyses the formation of a linear tetrapyrrole from four molecules of the substrate porphobilinogen (PBG). It has a dipyrromethane cofactor (DPM) in the active site which is covalently linked to a conserved cysteine residue through a thioether bridge. The substrate molecules are linked to the cofactor in a stepwise head-to-tail manner during the reaction, which is catalysed by a conserved aspartate residue: Asp82 in the B. megaterium enzyme. Three mutations have been made affecting Asp82 (D82A, D82E and D82N) and their crystal structures have been determined at resolutions of 2.7, 1.8 and 1.9 Å, respectively. These structures reveal that whilst the D82E mutant possesses the DPM cofactor, in the D82N and D82A mutants the cofactor is likely to be missing, incompletely assembled or disordered. Comparison of the mutant PBGD structures with that of the wild-type enzyme shows that there are significant domain movements and suggests that the enzyme adopts `open' and `closed' conformations, potentially in response to substrate binding

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

Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. Frutapin (FTP) is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically-active recombinant FTP in E. coli BL21, optimizing conditions with the best set yielding >40 mg/L culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/mL of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP-mannose and FTP-glucose crystals were obtained and diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å, respectively. The best solution showed four monomers per asymmetric unit. Molecular Dynamics simulation suggested FTP displays higher affinity for mannose than glucose. Cell studies revealed FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/mL and also capable of stimulating cell migration at 50 µg/mL. In conclusion, our optimized expression system allowed high amounts of correctly-folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example, in wound healing and tissue regeneration

Differences in Signaling Patterns on PI3K Inhibition Reveal Context Specificity in <i>KRAS</i>-Mutant Cancers.

It is increasingly appreciated that drug response to different cancers driven by the same oncogene is different and may relate to differences in rewiring of signal transduction. We aimed to study differences in dynamic signaling changes within mutant KRAS (KRAS MT), non-small cell lung cancer (NSCLC), colorectal cancer, and pancreatic ductal adenocarcinoma (PDAC) cells. We used an antibody-based phosphoproteomic platform to study changes in 50 phosphoproteins caused by seven targeted anticancer drugs in a panel of 30 KRAS MT cell lines and cancer cells isolated from 10 patients with KRAS MT cancers. We report for the first time significant differences in dynamic signaling between colorectal cancer and NSCLC cell lines exposed to clinically relevant equimolar concentrations of the pan-PI3K inhibitor pictilisib including a lack of reduction of p-AKTser473 in colorectal cancer cell lines (P = 0.037) and lack of compensatory increase in p-MEK in NSCLC cell lines (P = 0.036). Differences in rewiring of signal transduction between tumor types driven by KRAS MT cancers exist and influence response to combination therapy using targeted agents

Health System Resource Gaps and Associated Mortality from Pandemic Influenza across Six Asian Territories

BACKGROUND: Southeast Asia has been the focus of considerable investment in pandemic influenza preparedness. Given the wide variation in socio-economic conditions, health system capacity across the region is likely to impact to varying degrees on pandemic mitigation operations. We aimed to estimate and compare the resource gaps, and potential mortalities associated with those gaps, for responding to pandemic influenza within and between six territories in Asia. METHODS AND FINDINGS: We collected health system resource data from Cambodia, Indonesia (Jakarta and Bali), Lao PDR, Taiwan, Thailand and Vietnam. We applied a mathematical transmission model to simulate a "mild-to-moderate" pandemic influenza scenario to estimate resource needs, gaps, and attributable mortalities at province level within each territory. The results show that wide variations exist in resource capacities between and within the six territories, with substantial mortalities predicted as a result of resource gaps (referred to here as "avoidable" mortalities), particularly in poorer areas. Severe nationwide shortages of mechanical ventilators were estimated to be a major cause of avoidable mortalities in all territories except Taiwan. Other resources (oseltamivir, hospital beds and human resources) are inequitably distributed within countries. Estimates of resource gaps and avoidable mortalities were highly sensitive to model parameters defining the transmissibility and clinical severity of the pandemic scenario. However, geographic patterns observed within and across territories remained similar for the range of parameter values explored. CONCLUSIONS: The findings have important implications for where (both geographically and in terms of which resource types) investment is most needed, and the potential impact of resource mobilization for mitigating the disease burden of an influenza pandemic. Effective mobilization of resources across administrative boundaries could go some way towards minimizing avoidable deaths

Health plan administrative records versus birth certificate records: quality of race and ethnicity information in children

<p>Abstract</p> <p>Background</p> <p>To understand racial and ethnic disparities in health care utilization and their potential underlying causes, valid information on race and ethnicity is necessary. However, the validity of pediatric race and ethnicity information in administrative records from large integrated health care systems using electronic medical records is largely unknown.</p> <p>Methods</p> <p>Information on race and ethnicity of 325,810 children born between 1998-2008 was extracted from health plan administrative records and compared to birth certificate records. Positive predictive values (PPV) were calculated for correct classification of race and ethnicity in administrative records compared to birth certificate records.</p> <p>Results</p> <p>Misclassification of ethnicity and race in administrative records occurred in 23.1% and 33.6% children, respectively; the majority due to missing ethnicity (48.3%) and race (40.9%) information. Misclassification was most common in children of minority groups. PPV for White, Black, Asian/Pacific Islander, American Indian/Alaskan Native, multiple and other was 89.3%, 86.6%, 73.8%, 18.2%, 51.8% and 1.2%, respectively. PPV for Hispanic ethnicity was 95.6%. Racial and ethnic information improved with increasing number of medical visits. Subgroup analyses comparing racial classification between non-Hispanics and Hispanics showed White, Black and Asian race was more accurate among non-Hispanics than Hispanics.</p> <p>Conclusions</p> <p>In children, race and ethnicity information from administrative records has significant limitations in accurately identifying small minority groups. These results suggest that the quality of racial information obtained from administrative records may benefit from additional supplementation by birth certificate data.</p

'How to know what you need to do': a cross-country comparison of maternal health guidelines in Burkina Faso, Ghana and Tanzania

Initiatives to raise the quality of care provided to mothers need to be given priority in Sub Saharan Africa (SSA). The promotion of clinical practice guidelines (CPGs) is a common strategy, but their implementation is often challenging, limiting their potential impact. Through a cross-country perspective, this study explored CPGs for maternal health in Burkina Faso, Ghana, and Tanzania. The objectives were to compare factors related to CPG use including their content compared with World Health Organization (WHO) guidelines, their format, and their development processes. Perceptions of their availability and use in practice were also explored. The overall purpose was to further the understanding of how to increase CPGs' potential to improve quality of care for mothers in SSA. The study was a multiple case study design consisting of cross-country comparisons using document review and key informant interviews. A conceptual framework to aid analysis and discussion of results was developed, including selected domains related to guidelines' implementability and use by health workers in practice in terms of usability, applicability, and adaptability. The study revealed few significant differences in content between the national guidelines for maternal health and WHO recommendations. There were, however, marked variations in the format of CPGs between the three countries. Apart from the Ghanaian and one of the Tanzanian CPGs, the levels of both usability and applicability were assessed as low or medium. In all three countries, the use of CPGs by health workers in practice was perceived to be limited. Our cross-country study suggests that it is not poor quality of content or lack of evidence base that constitute the major barrier for CPGs to positively impact on quality improvement in maternal care in SSA. It rather emphasises the need to prioritise the format of guidelines to increase their usability and applicability and to consider these attributes together with implementation strategies as integral to their development processes