Evidence for a Dinuclear Active Site in the Metallo-β-lactamase BcII with Substoichiometric Co(II): A New Model for Uptake

Metallo-β-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to β-lactam antibiotics. Metallo-β-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-β-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His116, His118, and His196) and DCH (Asp120, Cys221, and His263) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-β-lactamase turnover

Asp-120 Locates Zn2 for Optimal Metallo-β-lactamase Activity

Metallo-β-lactamases are zinc-dependent hydrolases that inactivate β-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-β-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the β-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule

The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs

Trapping and Characterization of a Reaction Intermediate in Carbapenem Hydrolysis by \u3cem\u3eB. cereus\u3c/em\u3e Metallo-β-lactamase

Metallo-β-lactamases hydrolyze most β-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze−quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)−BcII with imipenem. These studies show that Co(II)−BcII is able to hydrolyze imipenem in both the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme−intermediate adduct in which the β-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonant structure that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-state-based inhibitors for all three classes of metallo-β-lactamases

Heart of Darkness: Heart Rate Variability on Patients with Risk of Suicide

Heart Rate Variability (HRV) is an emerging research field in the study of diverse pathologies, as long as it allows considering another measurement for detecting possible aggravations. The aim of this work is to study the applicability of the analysis of HRV in order to establish if a person is at risk of suffering from suicidal ideation. This work includes the development and testing of a heart rate acquisition and automatic analysis system, with friendly software for clinicians, customized to the necessities of an emergency unit. Furthermore, it includes the analysis of the obtained data with the purpose of assessing possible correlations between HRV parameters and personality impulsive traits. 20 patients and 10 normal cases were selected to develop this pilot study. Results show significant statistical difference (p<0.05) among patients and normal cases for pNN50, IRRR, MADRR, total HRV power, Approximate Entropy and Fractal Dimension

Author correction : A small Cretaceous crocodyliform in a dinosaur nesting ground and the origin of sebecids

Correction to: Scientific Reports https://doi.org/10.1038/s41598-020-71975-y, published online 17 September 202

Ph Salival como factor asociado a la caries dental

En la cavidad bucal existen múltiples factores que favorecen el desarrollo de caries dental. El presente estudio descriptivo, transversal tiene el propósito de conocer la relación entre las variaciones pH salival y presencia de caries en pacientes que asisten a la cátedra Práctica Clínica Preventiva II de la Facultad de Odontología. La población de estudio estuvo constituida por 30 adolescentes de entre 13 y 26 años de ambos sexos que concurrieron durante el periodo marzo a diciembre de 2011 para su atención. Se incluyeron aquellos clínicamente sanos, con estado nutricional normal y que no hayan consumido ningún tipo de alimento dos horas antes de la toma de muestra. Como criterios de exclusión se considero que posean enfermedades sistémicas, alteración en glándulas salivales y que puedan estar tomando alguna medicación anticolinérgica. A cada uno de ellos se le explico objetivos y métodos a utilizar verbalmente y escrito a través de un consentimiento informado, posteriormente se realizo una historia clínica completa , odontograma para establecer de esta manera el índice CPOD, consistente en observar las piezas dentarias careadas, perdidas y obturadas tomando como unidad el diente, siguiendo los criterios establecidos por la OMS. Subsiguientemente se coloco en piso de boca en contacto directo con la saliva la tira de prueba reactiva del pH se espero 5 minutos y se observo el cambio de color con el tiempo transcurrido. Finalmente se comparo el color de la ISSN Nº 1668-7280 - Vol. VI Nº 1 - 2013 REVISTA FACULTAD DE ODONTOLOGÍA 14 | INVESTIGACIÓN Rabaldo Bottan - Marin - Marchiori de Araújo REVISTA FACULTAD DE ODONTOLOGÍA 14 | INVESTIGACIÓN Barrios - Vila - Martínez - Encina Tutuy almohadilla de prueba con el cuadro de colores normal. Este indicador colorido refleja el pH de la tira. De acuerdo a las variables estudiadas, los resultados obtenidos en el índice de caries, son elevados no compatibles con salud, y en relación al pH la mayoría presenta un pH entre 6 y 7 manifestándose un alto índice de caries en personas que presentan un pH 5

A small Cretaceous crocodyliform in a dinosaur nesting ground and the origin of sebecids

Sebecosuchia was a group of highly specialized cursorial crocodyliforms that diversified during the Cretaceous and persist until the end of the Miocene. Their unique combination of cranial and post-cranial features indicates that they were active terrestrial predators that occupied the apex of the Late Cretaceous terrestrial ecosystems, even competing with theropod dinosaurs. Here, we report the discovery of the earliest sebecid worldwide, and the first from Eurasia, Ogresuchus furatus gen. et sp. nov., based on a semi-articulate specimen located in a titanosaurian sauropod nesting ground. The new taxon challenges current biogeographical models about the early dispersal and radiation of sebecid crocodylomorphs, and suggests an origin of the group much earlier than previously expected. Moreover, the new taxon suggests a potential convergent evolution between linages geographically isolated. Taphonomic evidences suggest that Ogresuchus died almost in the same place where fossilized, in a dinosaur nesting area. Biometric and morphologic observations lead to speculate that Ogresuchus could easily predate on sauropod hatchlings

Backbone Dynamics of Plastocyanin in Both Oxidation States SOLUTION STRUCTURE OF THE REDUCED FORM AND COMPARISON WITH THE OXIDIZED STATE

A model-free analysis based on15N R 1, 15NR 2, and 15N-1H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D2O exchange of amide protons and from NH-H2O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes