Co-Catalytic Metallopeptidases as Pharmaceutical Targets

Understanding the reaction mechanism of co-catalytic metallopeptidases provides a starting point for the design and synthesis of new molecules that can be screened as potential pharmaceuticals. Many of the enzymes that contain co-catalytic metallo-active sites play important roles in cellular processes such as tissue repair, protein maturation, hormone level regulation, cell-cycle control and protein degradation. Therefore, these enzymes play central roles in several disease states including cancer, HIV, stroke, diabetes, bacterial infections, neurological processes, schizophrenia, seizure disorders, and amyotrophic lateral sclerosis. The mechanism of AAP, an aminopeptidase from Aeromonas proteolytica, is one of the best-characterized examples of a metallopeptidase containing a co-catalytic metallo-active site, although this enzyme is not a specific pharmaceutical target at this time. As a large majority of co-catalytic metallopeptidases contain active sites that are nearly identical to the one observed in AAP, the major steps of their catalytic mechanisms are likely to be very similar. With this in mind, it is possible to propose a general catalytic mechanism for the hydrolysis of amino acid substrates

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

Effects of increased autumn temperatures and sub-acute levels of ammonia on post-winter development of four cultivars of winter oilseed rape (Brassica napus L.)

Early planting dates, warm autumns and increased N mineralisation  rates may advance the growth of winter crops and also a high supply of nitrogen poses risks of excess biomass accumulation before the winter leading to reduced frost hardiness and excessive leaf loss. Although the number of frost days is currently declining in temperate Europe, events of frost may still create damage in the future in winter crops that have accumulated too much biomass in the preceding autumn.Here we report on a study in which the responses of four cultivars of winter oilseed rape (OSR) to elevated autumn temperatures and subacute levels of ammonia (NH3) were compared. It has been suggested that high concentrations of the gas, e.g. in livestock intensive regions may both act as an airborne fertiliser and reduce the frost hardiness of plants. Advancing treatments were imposed for 88 days in the autumn and consisted of pot grown plants kept in greenhouses without (ELVT) and with elevated concentrations of ammonia (ELVT+A; 195 μg m-3 NH3). Reference plants (AMB) were raised outdoors, where temperatures were 1.4°C lower than in the greenhouses.After the treatments plants were all overwintered outside to study whether the pre-winter growth advancement was still discernible in the following spring. Shed leaves were collected weekly to follow how much shoot biomass was lost during and after the winter. Cultivars responded differently to warming and exposure to ammonia. Up to the winter shoot biomass was strongly increased by the advancing treatments. However, fi nal shoot mass in the following summer did not differ between cultivars and was unaffected by the higher temperatures in the preceding autumn. Nevertheless, significantly more biomass was observed in ammonia fumigated plants. Higher autumn temperatures increased leaf shedding and advanced fl owering and senescence in the next spring so that plants showed a signifi cantly reduced seed mass, harvest index and oil yield at the fi nal harvest. Obviously, the growth advancement in the preceding autumn by elevated temperatures negatively affected the availability of resources in the following spring. In contrast, plants that were grown at both elevated ammonia and temperature in the autumn showed a delayed fl owering, higher shoot and seed mass, increased harvest index and oil yield. We conclude that growth advancement by elevated autumn temperatures without the re-supply of nutrients increases leaf shedding during winter. Nevertheless, the loss of resources in winter for re-growth in spring will certainly be of minor importance for yield formation as compared to the frost damage resulting from late spring frosts

Double Compact Objects III: Gravitational Wave Detection Rates

The unprecedented range of second-generation gravitational-wave (GW) observatories calls for refining the predictions of potential sources and detection rates. The coalescence of double compact objects (DCOs)---i.e., neutron star-neutron star (NS-NS), black hole-neutron star (BH-NS), and black hole-black hole (BH-BH) binary systems---is the most promising source of GWs for these detectors. We compute detection rates of coalescing DCOs in second-generation GW detectors using the latest models for their cosmological evolution, and implementing inspiral-merger-ringdown (IMR) gravitational waveform models in our signal-to-noise ratio calculations. We find that: (1) the inclusion of the merger/ringdown portion of the signal does not significantly affect rates for NS-NS and BH-NS systems, but it boosts rates by a factor $\sim 1.5$ for BH-BH systems; (2) in almost all of our models BH-BH systems yield by far the largest rates, followed by NS-NS and BH-NS systems, respectively, and (3) a majority of the detectable BH-BH systems were formed in the early Universe in low-metallicity environments. We make predictions for the distributions of detected binaries and discuss what the first GW detections will teach us about the astrophysics underlying binary formation and evolution.Comment: published in ApJ, 19 pages, 11 figure

Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in kcat. The kcat value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while kcat decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The Km values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The kcat/Km values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from ∼50- to 580-fold reduction. The pH dependence of log Km, log kcat, and log(kcat/Km) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pKa values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at λmax(640) vs pH for both WT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site

Phylogeny, character evolution, and biogeography of the gondwanic moss family Hypopterygiaceae (Bryophyta)

Phylogenetic relationships among the seven genera of the Hypopterygiaceae, represented by 14 of the 21 species recognized in the family, were reconstructed based on variation in nucleotide sequences of six nuclear, mitochondrial, and plastid loci

The last glacial termination on the eastern flank of the central Patagonian Andes (47ºS)

Few studies have examined in detail the sequence of events during the last glacial termination (T1) in the core sector of the Patagonian Ice Sheet (PIS), the largest ice mass in the Southern Hemisphere outside of Antarctica. Here we report results from Lago Edita (47°8′ S, 72°25′ W, 570 m a.s.l.), a small closed-basin lake located in a valley overridden by eastward-flowing Andean glaciers during the Last Glacial Maximum (LGM). The Lago Edita record shows glaciolacustrine sedimentation until 19 400 yr BP, followed by organic sedimentation in a closed-basin lake and a mosaic of cold-resistant hygrophilous conifers and rainforest trees, along with alpine herbs between 19 400 and 11 000 yr BP. Our data suggest that the PIS retreated at least  ∼  90 km from its LGM limit between  ∼  21 000 and 19 400 yr BP and that scattered, low-density populations of cold-resistant hygrophilous conifers, rainforest trees, high-Andean and steppe herbs thrived east of the Andes during the LGM and T1, implying high precipitation levels and southern westerly wind (SWW) influence at 47° S. The conifer Podocarpus nubigena increased between 14 500 and 13 000 yr BP, suggesting even stronger SWW influence during the Antarctic Cold Reversal, after which it declined and persisted until 11 000 yr BP. Large increases in arboreal pollen at  ∼  13 000 and  ∼  11 000 yr BP led to the establishment of forests near Lago Edita between 10 000 and 9000 yr BP, suggesting a rise in the regional tree line along the eastern Andean slopes driven by warming pulses at  ∼  13 000 and  ∼  11 000 yr BP and a subsequent decline in SWW influence at  ∼  11 000 yr BP. We propose that the PIS imposed a regional cooling signal along its eastern, downwind margin through T1 that lasted until the separation of the northern and southern Patagonian ice fields along the Andes during the Younger Dryas period. We posit that the withdrawal of glacial and associated glaciolacustrine environments through T1 provided a route for the dispersal of hygrophilous trees and herbs from the eastern flank of the central Patagonian Andes, contributing to the afforestation of the western Andean slopes and pacific coasts of central Patagonia during T1.Fil: Henríquez, William I.. Victoria University Of Wellington; Nueva ZelandaFil: Villa-Martinez, Rodrigo. Gaia-antártica Universidad de Magallanes; ChileFil: Vilanova, Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales ; ArgentinaFil: De Pol-Holz, Ricardo. Gaia-antártica Universidad de Magallanes; ChileFil: Moreno, Patricio. Instituto de Ecología y Biodiversidad, Departamento de; Chil

Spectroscopic and Thermodynamic Characterization of the E151D and E151A Altered Leucine Aminopeptidases from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 Å in carboxypeptidase G2 and 3.30 and 3.63 Å in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by UV−vis and electron paramagnetic resonance (EPR) spectroscopy. UV−vis spectroscopy of Co(II)-substituted E151A-AAP did not reveal any significant changes in the electronic absorption spectra. However UV−vis spectra of mono- and dicobalt(II) E151D-AAP exhibited a lower molecular absorptivity compared to AAP (23 and 43 M-1 cm-1 vs. 56 and 109 M-1 cm-1 for E151D-AAP and AAP, respectively) suggesting both Co(II) ions reside in distorted octahedral coordination geometry in E151D-AAP. EPR spectra of [Co_(E151D-AAP)], [ZnCo(E151D-AAP)], and [(CoCo(E151D-AAP)] were identical, with g⊥ = 2.35, g∥ = 2.19, and E/D = 0.19, similar to [CoCo(AAP)]. On the other hand, the EPR spectrum of [Co_(E151A-AAP)] was best simulated assuming the presence of two species with (i) gx,y = 2.509, gz = 2.19, E/D = 0.19, A = 0.0069 cm-1 and (ii) gx,y = 2.565, gz = 2.19, E/D = 0.20, A = 0.0082 cm-1 indicative of a five- or six-coordinate species. Isothermal titration calorimetry experiments revealed a large decrease in Zn(II) affinities, with Kd values elevated by factors of ∼850 and ∼24 000 for the first metal binding events of E151D- and E151A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP