Characterization of the Catalytically Active Mn(II)-loaded \u3cem\u3eargE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-acetyl-L-ornithine Deacetylase from \u3cem\u3eEscherichia coli\u3c/em\u3e

The catalytically competent Mn(II)-loaded form of the argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli (ArgE) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N-acetyl-l-ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k cat and K m values of 550 s−1 and 0.8 mM, respectively, providing a catalytic efficiency (k cat/K m) of 6.9 × 105 M−1 s−1. The ArgE dissociation constant (K d) for Mn(II) was determined to be 0.18 μM, correlating well with a value obtained by isothermal titration calorimetry of 0.30 μM for the first metal binding event and 5.3 μM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded ArgE was linear from 15 to 55 °C, suggesting the rate-limiting step does not change as a function of temperature over this range. The activation energy, determined from the slope of this plot, was 50.3 kJ mol−1. Other thermodynamic parameters were ΔG ‡ = 58.1 kJ mol−1, ΔH ‡ = 47.7 kJ mol−1, and ΔS ‡ = –34.5 J mol−1 K−1. Similarly, plots of lnK m versus 1/T were linear, suggesting substrate binding is controlled by a single step. The natural product, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]leucine (bestatin), was found to be a competitive inhibitor of ArgE with a K i value of 67 μM. Electron paramagnetic resonance (EPR) data recorded for both [Mn(II)_(ArgE)] and [Mn(II)Mn(II)(ArgE)] indicate that the two Mn(II) ions form a dinuclear site. Moreover, the EPR spectrum of [Mn(II)Mn(II)(ArgE)] in the presence of bestatin indicates that bestatin binds to ArgE but does not form a µ-alkoxide bridge between the two metal ions

\u3cem\u3eargE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Acetyl-l-Ornithine Deacetylase from \u3cem\u3eEscherichia coli\u3c/em\u3e Contains a Dinuclear Metalloactive Site

The catalytic and structural properties of the argE-encoded N-acetyl-l-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by ∼20 times. Moreover, ArgE exhibits ∼90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-l-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 °C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co·(ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin−spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2)

Identification of a Histidine Metal Ligand in the \u3cem\u3eargE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Acetyl-L-Ornithine Deacetylase from \u3cem\u3eEscherichia coli\u3c/em\u3e

The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (Kd) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands

Grid-connected renewables, storage and the UK electricity market

This article is a critical counterpoint to an article by published by Swift-Hook in the journal of Renewable Energy entitled "Grid-connected intermittent renewables are the last to be stored". In contrast to Swift-Hook we found evidence that "grid-connected intermittent renewables" have been, and will continue to be stored when it suits the "UK market" to do so.  This article is important to policy makers as energy storage (through EV battery demand side management for example) may well have an important role to play in facilitating the integration of high wind penetrations

The voices of local NGOs in climate change issues: Examples from climate vulnerable nations

© 2018 by the author(s). The contributions of small local non-government organisations (NGOs) in countries at risk from climate change to knowledge creation and action on climate change are rarely considered. This study sought to remedy this by focusing on NGOs in member countries of the Climate Vulnerable Forum (CVF). Analysing data from Intended Nationally Determined Contributions (INDCs), NGO websites and email correspondence with NGO staff through a knowledge brokering typology, this study examines the ways in which local NGOs in five members of the CVF (Afghanistan, Bhutan, Kiribati, Nepal and Tuvalu) take action, generate new knowledge and understandings and contribute to the plans and actions of their government and the international community. The study found that local NGOs are involved in the creation of new knowledge both at the scientific and community level and engage in actions to support adaptation to climate change. However, there are differences in the approaches they take when making contributions to scientific knowledge and climate change debates. The findings of this study suggest the need to reconceptualise the role of local NGOs in small countries at risk from climate change

Characterization of rutile passivation layers formed on Magnéli-phase titanium oxide inert anodes

An ex situ characterization study has been performed on rutile passivation layers on inert anodes used for molten salt electrochemical studies. Rutile layer thicknesses were estimated using a number of ex situ methods, including laboratory and synchrotron X-ray diffraction and optical microscopy. The only phases in the anode detected by diffraction were the Magnéli phases (TinO2n-1, n = 5-6) of the unreacted anode and rutile (TiO2), which forms on electrolysis. These measurements validate a previously developed in situ energy-dispersive X-ray diffraction analysis technique [Scarlett, Madsen, Evans, Coelho, McGregor, Rowles, Lanyon & Urban (2009). J. Appl. Cryst. 42, 502-512]

Pattern matching and pattern discovery algorithms for protein topologies

We describe algorithms for pattern matching and pattern learning in TOPS diagrams (formal descriptions of protein topologies). These problems can be reduced to checking for subgraph isomorphism and finding maximal common subgraphs in a restricted class of ordered graphs. We have developed a subgraph isomorphism algorithm for ordered graphs, which performs well on the given set of data. The maximal common subgraph problem then is solved by repeated subgraph extension and checking for isomorphisms. Despite the apparent inefficiency such approach gives an algorithm with time complexity proportional to the number of graphs in the input set and is still practical on the given set of data. As a result we obtain fast methods which can be used for building a database of protein topological motifs, and for the comparison of a given protein of known secondary structure against a motif database