Crystal structure of Pseudomonas aeruginosa lipase in the open conformation - The prototype for family I.1 of bacterial lipases

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 Angstrom resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two P-strands and one alpha-helix (alpha E) are not present. The absence of helix alpha E allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His251 is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme

The complexities of electronic services implementation and institutionalisation in the public sector

This is the post-print version of the final paper published in Information & Management. The published article is available from the link below. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. Copyright @ 2013 Elsevier B.V.Electronic service implementation (ESI) in the public sector attempts to improve efficiency, effectiveness, and transparency of governmental departments. Despite having provided the necessary infrastructure and investment, many governments have struggled to realise such aims due to the various forces that challenge implementation and institutionalisation. Using institutional theory as a lens, we explored the forces influencing the implementation and institutionalisation of ESI in the public sector. While our results reinforced previous research in IT implementation and organisational transformation, they showed that the dynamic nature of technology poses unanticipated pressures, and that these can impede the implementation and institutionalisation process

Directed evolution of an enantioselective Bacillus subtilis lipase

Chiral compounds are of steadily increasing importance to the chemical industry, in particular for the production of pharmaceuticals. Where do these compounds come from? Apart from natural resources, two synthetic strategies are available: asymmetric chemical catalysis using transition metal catalysts and biocatalysis using enzymes. In the latter case, screening programs have identified a number of enzymes. However, their enantioselectivity is often not high enough for a desired reaction. This problem can be solved by applying directed evolution to create enantioselective enzymes as shown here for a lipase from Bacillus subtilis. The reaction studied was the asymmetric hydrolysis of meso-1,4-diacetoxy-2-cyclopentene with the formation of chiral alcohols which were detected by electrospray ionization mass spectrometry. Iterative cycles of random mutagenesis and screening allowed the identification of several variants with improved enantioselectivities. In parallel, we have started to use X-ray structural data to simulate the Bacillus subtilis lipase A-catalyzed substrate hydrolysis by using quantum mechanical and molecular mechanical calculations. This combined approach should finally enable us to devise more efficient strategies for the directed evolution of enantioselective enzymes

Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

Background: Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings: A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance: The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity

The logic of the floral transition: reverse-engineering the switch controlling the identity of lateral organs

Much laboratory work has been carried out to determine the gene regulatory network (GRN) that results in plant cells becoming flowers instead of leaves. However, this also involves the spatial distribution of different cell types, and poses the question of whether alternative networks could produce the same set of observed results. This issue has been addressed here through a survey of the published intercellular distribution of expressed regulatory genes and techniques both developed and applied to Boolean network models. This has uncovered a large number of models which are compatible with the currently available data. An exhaustive exploration had some success but proved to be unfeasible due to the massive number of alternative models, so genetic programming algorithms have also been employed. This approach allows exploration on the basis of both data-fitting criteria and parsimony of the regulatory processes, ruling out biologically unrealistic mechanisms. One of the conclusions is that, despite the multiplicity of acceptable models, an overall structure dominates, with differences mostly in alternative fine-grained regulatory interactions. The overall structure confirms the known interactions, including some that were not present in the training set, showing that current data are sufficient to determine the overall structure of the GRN. The model stresses the importance of relative spatial location, through explicit references to this aspect. This approach also provides a quantitative indication of how likely some regulatory interactions might be, and can be applied to the study of other developmental transitions

Isolation and Characterization of EstC, a New Cold-Active Esterase from Streptomyces coelicolor A3(2)

The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5–9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6–11. The enzyme was active toward short-chain p-nitrophenyl esters (C2–C12), displaying optimal activity with the valerate (C5) ester (kcat/Km = 737±77 s−1 mM−1). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg[superscript 2+], Ca[superscript 2+], and Mn[superscript 2+] were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.Malaysia-MIT Biotechnology Partnership Programm

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines

Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1–29)NH2 re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1–29)NH2 and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor

The emerging structure of the Extended Evolutionary Synthesis: where does Evo-Devo fit in?

The Extended Evolutionary Synthesis (EES) debate is gaining ground in contemporary evolutionary biology. In parallel, a number of philosophical standpoints have emerged in an attempt to clarify what exactly is represented by the EES. For Massimo Pigliucci, we are in the wake of the newest instantiation of a persisting Kuhnian paradigm; in contrast, Telmo Pievani has contended that the transition to an EES could be best represented as a progressive reformation of a prior Lakatosian scientific research program, with the extension of its Neo-Darwinian core and the addition of a brand-new protective belt of assumptions and auxiliary hypotheses. Here, we argue that those philosophical vantage points are not the only ways to interpret what current proposals to ‘extend’ the Modern Synthesis-derived ‘standard evolutionary theory’ (SET) entail in terms of theoretical change in evolutionary biology. We specifically propose the image of the emergent EES as a vast network of models and interweaved representations that, instantiated in diverse practices, are connected and related in multiple ways. Under that assumption, the EES could be articulated around a paraconsistent network of evolutionary theories (including some elements of the SET), as well as models, practices and representation systems of contemporary evolutionary biology, with edges and nodes that change their position and centrality as a consequence of the co-construction and stabilization of facts and historical discussions revolving around the epistemic goals of this area of the life sciences. We then critically examine the purported structure of the EES—published by Laland and collaborators in 2015—in light of our own network-based proposal. Finally, we consider which epistemic units of Evo-Devo are present or still missing from the EES, in preparation for further analyses of the topic of explanatory integration in this conceptual framework