The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop

Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.Fil: Palacios, Antonela Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mojica, María F.. Case Western Reserve University; Estados UnidosFil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Alzari, Pedro M.. Institut Pasteur de Paris; FranciaFil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bonomo, Robert A.. Case Western Reserve University; Estados UnidosFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Sustainable bioethanol production combining biorefinery principles using combined raw materials from wheat undersown with clover-grass

To obtain the best possible net energy balance of the bioethanol production the biomass raw materials used need to be produced with limited use of non-renewable fossil fuels. Intercropping strategies are known to maximize growth and productivity by including more than one species in the crop stand, very often with legumes as one of the components. In the present study clover-grass is undersown in a traditional wheat crop. Thereby, it is possible to increase input of symbiotic fixation of atmospheric nitrogen into the cropping systems and reduce the need for fertilizer applications. Furthermore, when using such wheat and clover-grass mixtures as raw material, addition of urea and other fermentation nutrients produced from fossil fuels can be reduced in the whole ethanol manufacturing chain. Using second generation ethanol technology mixtures of relative proportions of wheat straw and clover-grass (15:85, 50:50, and 85:15) were pretreated by wet oxidation. The results showed that supplementing wheat straw with clover-grass had a positive effect on the ethanol yield in simultaneous saccharification and fermentation experiments, and the effect was more pronounced in inhibitory substrates. The highest ethanol yield (80% of theoretical) was obtained in the experiment with high fraction (85%) of clover-grass. In order to improve the sugar recovery of clover-grass, it should be separated into a green juice (containing free sugars, fructan, amino acids, vitamins and soluble minerals) for direct fermentation and a fibre pulp for pretreatment together with wheat straw. Based on the obtained results a decentralized biorefinery concept for production of biofuel is suggested emphasizing sustainability, localness, and recycling principle

Horizon effects with surface waves on moving water

Surface waves on a stationary flow of water are considered, in a linear model that includes the surface tension of the fluid. The resulting gravity-capillary waves experience a rich array of horizon effects when propagating against the flow. In some cases three horizons (points where the group velocity of the wave reverses) exist for waves with a single laboratory frequency. Some of these effects are familiar in fluid mechanics under the name of wave blocking, but other aspects, in particular waves with negative co-moving frequency and the Hawking effect, were overlooked until surface waves were investigated as examples of analogue gravity [Sch\"utzhold R and Unruh W G 2002 Phys. Rev. D 66 044019]. A comprehensive presentation of the various horizon effects for gravity-capillary waves is given, with emphasis on the deep water/short wavelength case kh>>1 where many analytical results can be derived. A similarity of the state space of the waves to that of a thermodynamic system is pointed out.Comment: 30 pages, 15 figures. Minor change

Radial elasticity of multi-walled carbon nanotubes

We report an experimental and a theoretical study of the radial elasticity of multi-walled carbon nanotubes as a function of external radius. We use atomic force microscopy and apply small indentation amplitudes in order to stay in the linear elasticity regime. The number of layers for a given tube radius is inferred from transmission electron microscopy, revealing constant ratios of external to internal radii. This enables a comparison with molecular dynamics results, which also shed some light onto the applicability of Hertz theory in this context. Using this theory, we find a radial Young modulus strongly decreasing with increasing radius and reaching an asymptotic value of 30 +/- 10 GPa.Comment: 5 pages, 3 figure

Microbreaking and the enhancement of air-water transfer velocity

The role of microscale wave breaking in controlling the air-water transfer of heat and gas is investigated in a laboratory wind-wave tank. The local heat transfer velocity, kH, is measured using an active infrared technique and the tank-averaged gas transfer velocity, kG, is measured using conservative mass balances. Simultaneous, colocated infrared and wave slope imagery show that wave-related areas of thermal boundary layer disruption and renewal are the turbulent wakes of microscale breaking waves, or microbreakers. The fractional area coverage of microbreakers, AB, is found to be 0.1–0.4 in the wind speed range 4.2–9.3 m s−1 for cleaned and surfactant-influenced surfaces, and kH and kG are correlated with AB. The correlation of kH with AB is independent of fetch and the presence of surfactants, while that for kG with AB depends on surfactants. Additionally, AB is correlated with the mean square wave slope, 〈S2〉, which has shown promise as a correlate for kG in previous studies. The ratio of kH measured inside and outside the microbreaker wakes is 3.4, demonstrating that at these wind speeds, up to 75% of the transfer is the direct result of microbreaking. These results provide quantitative evidence that microbreaking is the dominant mechanism contributing to air-water heat and gas transfer at low to moderate wind speeds

Algorithm for multi-curve-fitting with shared parameters and a possible application in evoked compound action potential measurements

BACKGROUND: Experimental results are commonly fitted by determining parameter values of suitable mathematical expressions. In case a relation exists between different data sets, the accuracy of the parameters obtained can be increased by incorporating this relationship in the fitting process instead of fitting the recordings separately. METHODS: An algorithm to fit multiple measured curves simultaneously was developed. The method accounts for parameters that are shared by some curves. It can be applied to either linear or nonlinear equations. Simulated noisy "measurement results" were created to compare the introduced method to the "straight forward" way of fitting the curves separately. RESULTS: The analysis of the simulated measurements confirm, that the introduced method yields more accurate parameters compared to the ones gained by fitting the measurements separately. Therefore it needs more computer time. As an example, the new fitting algorithm is applied to the measurements of the evoked compound action potentials (ECAP) of the auditory nerve: This leads to promising ideas to reduce artefacts generated by the measuring process. CONCLUSION: The introduced fitting algorithm uses the relationship between multiple measurement results to increase the accuracy of the parameters. Its application in the field of ECAP measurements is promising and should be further investigated

Field-effect transistors assembled from functionalized carbon nanotubes

We have fabricated field effect transistors from carbon nanotubes using a novel selective placement scheme. We use carbon nanotubes that are covalently bound to molecules containing hydroxamic acid functionality. The functionalized nanotubes bind strongly to basic metal oxide surfaces, but not to silicon dioxide. Upon annealing, the functionalization is removed, restoring the electronic properties of the nanotubes. The devices we have fabricated show excellent electrical characteristics.Comment: 5 pages, 6 figure