Structural insights into TMB-1 and the role of residues 119 and 228 in substrate and inhibitor binding

Source at https://doi.org/10.1128/AAC.02602-16.Metallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 μM (inhibitor 2a) and 0.62 μM (inhibitor 2b), and the equilibrium dissociation constant (KD) of inhibitor 2a was 1.6 μM; thus, both were more potent inhibitors than L-captopril (IC50 = 47 μM; KD = 25 μM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than L-captopril

Seasonal and interannual variability in abundance and population development of Calanus finmarchicus at the western entrance to the Barents Sea, 1995–2019

Data on copepodid abundance and stage composition of Calanus finmarchicus was collected with seasonal resolution (5–6 times a year) from four stations along an oceanographic transect across the western Barents Sea Opening over 25 years (1995–2019). The stations were located in the Norwegian Coastal Current (NCC), inflowing Atlantic water (two stations), and near the Arctic polar front south of Bear Island. Mean copepodite abundance in the water column for the four stations increased from a level around 1000–10,000 individuals m−2 in winter to 30,000–100,000 individuals m−2 in summer (maximum 670,000 m−2). The overwintering (G0) population was dominated by copepodite stage 5 (CV) (40–70%) but with a relatively high fraction also of stage CIV (25–35%) in January. During winter, there was a progressive development of CVs into adult males and females, with mean abundances from 1500 to 4000 adult females m−2 in April and May for the stations in the NCC and Atlantic water. Young copepodids of the new spring generation (G1) appeared with relatively high abundances in April at the NCC station and in May at the Atlantic water stations. The data showed a gradient of later development from south to north, reflected both in the maturation of the overwintering generation and in the development of the spring generation. Back-calculations based on temperature-dependent development time suggested peak spawning around late April to mid-May for the Atlantic water stations, and a month or so earlier (in March) for the NCC station, during an early phase of the spring phytoplankton growth. The spring generation developed as a distinct cohort but with large interannual variability in numbers. In Atlantic water, the composition of the copepodid developmental stages in August resembled the situation in June, although it was slightly more advanced. This is interpreted to reflect development of a second generation (G2) spawned upstream sometime in June or early July. The June data suggests that only a small fraction of the G1 cohort develops into adults that spawn and form the G2. There was a tendency of increased summer abundance of C. finmarchicus in the inflow region of the southwestern Barents Sea after about 2005. This may be related to the wind regime with more eastward wind-stress at the Barents Sea opening in recent ‘high Calanus’ years compared to ‘low Calanus’ years in the early 2000s.publishedVersio

Fluctuating Atlantic inflows modulate Arctic atlantification

Enhanced warm, salty subarctic inflows drive high-latitude atlantification, which weakens oceanic stratification, amplifies heat fluxes, and reduces sea ice. In this work, we show that the atmospheric Arctic Dipole (AD) associated with anticyclonic winds over North America and cyclonic winds over Eurasia modulates inflows from the North Atlantic across the Nordic Seas. The alternating AD phases create a “switchgear mechanism.” From 2007 to 2021, this switchgear mechanism weakened northward inflows and enhanced sea-ice export across Fram Strait and increased inflows throughout the Barents Sea. By favoring stronger Arctic Ocean circulation, transferring freshwater into the Amerasian Basin, boosting stratification, and lowering oceanic heat fluxes there after 2007, AD+ contributed to slowing sea-ice loss. A transition to an AD− phase may accelerate the Arctic sea-ice decline, which would further change the Arctic climate system.acceptedVersio

Climate variability in the Barents Sea 2000-2005

Presented at the ICES Annual Science Conference, Maastricht, the Netherlands, 19–23 September 200

Wind-Driven Atlantic Water Flow as a Direct Mode for Reduced Barents Sea Ice Cover

Variability in the Barents Sea ice cover on interannual and longer time scales has previously been shown to be governed by oceanic heat transport. Based on analysis of observations and results from an ocean circulation model during an event of reduced sea ice cover in the northeastern Barents Sea in winter 1993, it is shown that the ocean also plays a direct role within seasons. Positive wind stress curl and associated Ekman divergence causes a coherent increase in the Atlantic water transport along the negative thermal gradient through the Barents Sea. The immediate response connected to the associated local winds in the northeastern Barents Sea is a decrease in the sea ice cover due to advection. Despite a subsequent anomalous ocean-to-air heat loss on the order of 100 W m22 due to the open water, the increase in the ocean heat content caused by the circulation anomaly reduced refreezing on a time scale of order one month. Furthermore, it is found that coherent ocean heat transport anomalies occurred more frequently in the latter part of the last five decades during periods of positive North Atlantic Oscillation index, coinciding with the Barents Sea winter sea ice cover decline from the 1990s and onward.publishedVersio

His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo beta Lactamase VIM 7

Metallo-β-lactamases (MBLs) are the causative mechanism for resistance to β-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T(m)) of 48.5°C, compared to 55.3°C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher T(m) (57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T(m), 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime

Closing the loop Approaches to monitoring the state of the Arctic Mediterranean during the International Polar Year 20072008

During the 4th International Polar Year 2007–2009 (IPY), it has become increasingly obvious that we need to prepare for a new era in the Arctic. IPY occurred during the time of the largest retreat of Arctic sea ice since satellite observations started in 1979. This minimum in September sea ice coverage was accompanied by other signs of a changing Arctic, including the unexpectedly rapid transpolar drift of the Tara schooner, a general thinning of Arctic sea ice and a double-dip minimum of the Arctic Oscillation at the end of 2009. Thanks to the lucky timing of the IPY, those recent phenomena are well documented as they have been scrutinized by the international research community, taking advantage of the dedicated observing systems that were deployed during IPY. However, understanding changes in the Arctic System likely requires monitoring over decades, not years. Many IPY projects have contributed to the pilot phase of a future, sustained, observing system for the Arctic. We now know that many of the technical challenges can be overcome. The Norwegian projects iAOOS-Norway, POLEWARD and MEOP were significant ocean monitoring/research contributions during the IPY. A large variety of techniques were used in these programs, ranging from oceanographic cruises to animal-borne platforms, autonomous gliders, helicopter surveys, surface drifters and current meter arrays. Our research approach was interdisciplinary from the outset, merging ocean dynamics, hydrography, biology, sea ice studies, as well as forecasting. The datasets are tremendously rich, and they will surely yield numerous findings in the years to come. Here, we present a status report at the end of the official period for IPY. Highlights of the research include: a quantification of the Meridional Overturning Circulation in the Nordic Seas (“the loop”) in thermal space, based on a set of up to 15-year-long series of current measurements; a detailed map of the surface circulation as well as characterization of eddy dispersion based on drifter data; transport monitoring of Atlantic Water using gliders; a view of the water mass exchanges in the Norwegian Atlantic Current from both Eulerian and Lagrangian data; an integrated physical–biological view of the ice-influenced ecosystem in the East Greenland Current, showing for instance nutrient-limited primary production as a consequence of decreasing ice cover for larger regions of the Arctic Ocean. Our sea ice studies show that the albedo of snow on ice is lower when snow cover is thinner and suggest that reductions in sea ice thickness, without changes in sea ice extent, will have a significant impact on the arctic atmosphere. We present up-to-date freshwater transport numbers for the East Greenland Current in the Fram Strait, as well as the first map of the annual cycle of freshwater layer thickness in the East Greenland Current along the east coast of Greenland, from data obtained by CTDs mounted on seals that traveled back and forth across the Nordic Seas. We have taken advantage of the real-time transmission of some of these platforms and demonstrate the use of ice-tethered profilers in validating satellite products of sea ice motion, as well as the use of Seagliders in validating ocean forecasts, and we present a sea ice drift product – significantly improved both in space and time – for use in operational ice-forecasting applications. We consider real-time acquisition of data from the ocean interior to be a vital component of a sustained Arctic Ocean Observing System, and we conclude by presenting an outline for an observing system for the European sector of the Arctic Ocean