RESUMEN
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCE: Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.
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Bacteriocinas , Cisteína , Tiazoles , Tiazoles/metabolismo , Cisteína/metabolismo , Bacteriocinas/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Procesamiento Proteico-Postraduccional , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.
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Proteínas Bacterianas/metabolismo , Bacteriocinas/metabolismo , Péptidos/metabolismo , Staphylococcaceae/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriocinas/química , Bacteriocinas/genética , Vías Biosintéticas/genética , Modelos Moleculares , Estructura Molecular , Péptidos/química , Péptidos/genética , Conformación Proteica , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcaceae/enzimología , Staphylococcaceae/genéticaRESUMEN
UNLABELLED: Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCE: The emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics.
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Bacillus subtilis/metabolismo , Bacteriocinas/biosíntesis , Regulación Bacteriana de la Expresión Génica/fisiología , Bacillus subtilis/genética , Bacteriocinas/química , Bacteriocinas/genética , Regulación Enzimológica de la Expresión Génica , Estructura Molecular , Familia de Multigenes , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Péptidos/química , Péptidos/genéticaRESUMEN
OBJECTIVE: Our study objectives were to determine the key sources of moral distress in diverse critical care professionals and how they manage it in the context of team-based models. DESIGN: Qualitative case study methodology using three recently resolved clinical cases. SETTING: A medical and surgical adult ICU in a 900-bed academic, tertiary Houston hospital. SUBJECTS: Twenty-nine ICU team members of diverse professional backgrounds interviewed between March 2013 and July 2013. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: All members of the ICU team reported experiencing moral distress. Intrateam discordance served as a key source of distress for all healthcare disciplines. Interviewees identified two situations where intrateam discordance creates moral distress: 1) situations involving initiation or maintenance of nonbeneficial life-sustaining treatments and 2) situations involving a lack of full disclosure about interventions. Healthcare professionals engaged in a variety of management techniques, which can be grouped according to maladaptive behaviors (pas-de-deux, "fighting," and withdrawing) and constructive behaviors (venting, mentoring networks, and building team cohesion). Maladaptive behaviors were more common in the surgical ICU. Constructive behaviors were more prevalent in the medical ICU and typically used by nurses and ancillary staff members. Physicians report becoming detached as morally distressing cases unfold, whereas nurses report becoming more emotionally invested. CONCLUSIONS: This study identified the ways in which moral distress manifests across critical care disciplines in different ICU environments. Our results have potential implications for patient care. First, when clinicians alter the content of their goals-of-care conversations with patients or families to accommodate intrateam discordance (as part of the "pas-de-deux"), subsequent decisions regarding medical care may be compromised. Second, when different team members respond differently to the same case-with nurses becoming more emotionally invested and physicians becoming more withdrawn-communication gaps are likely to occur at critical moral distress junctures. Finally, our findings suggest that physicians and any healthcare professionals in surgical units might be susceptible to unmitigated moral distress because they report less engagement in constructive behaviors to recalibrate their distress.
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Cuidados Críticos/psicología , Procesos de Grupo , Grupo de Atención al Paciente , Disentimientos y Disputas , Femenino , Humanos , Relaciones Interprofesionales , Entrevista Psicológica , Masculino , Relaciones Profesional-FamiliaRESUMEN
BACKGROUND: As next generation sequencing for the genetic diagnosis of cardiovascular disorders becomes more widely used, establishing causality for putative disease causing variants becomes increasingly relevant. Diseases of the cardiac sarcomere provide a particular challenge in this regard because of the complexity of assaying the effect of genetic variants in human cardiac contractile proteins. RESULTS: In this study we identified a novel variant R205Q in the cardiac troponin T gene (TNNT2). Carriers of the variant allele exhibited increased chamber volumes associated with decreased left ventricular ejection fraction. To clarify the causal role of this variant, we generated recombinant variant human protein and examined its calcium kinetics as well as the maximally activated ADP release of human ß-cardiac myosin with regulated thin filaments containing the mutant troponin T. We found that the R205Q mutation significantly decreased the calcium sensitivity of the thin filament by altering the effective calcium dissociation kinetics. CONCLUSIONS: The development of moderate throughput post-genomic assays is an essential step in the realization of the potential of next generation sequencing. Although technically challenging, biochemical and functional assays of human cardiac contractile proteins of the thin filament can be achieved and provide an orthogonal source of information to inform the question of causality for individual variants.
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Calcio/metabolismo , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/fisiopatología , Mutación , Troponina T/genética , Troponina T/metabolismo , Adulto , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Humanos , Técnicas In Vitro , Masculino , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Volumen SistólicoRESUMEN
The protean chemical properties of the toxic metal mercury (Hg) have made it attractive in diverse applications since antiquity. However, growing public concern has led to an international agreement to decrease its impact on health and the environment. During a recent proteomics study of acute Hg exposure in E. coli, we also examined the effects of inorganic and organic Hg compounds on thiol and metal homeostases. On brief exposure, lower concentrations of divalent inorganic mercury Hg(II) blocked bulk cellular thiols and protein-associated thiols more completely than higher concentrations of monovalent organomercurials, phenylmercuric acetate (PMA) and merthiolate (MT). Cells bound Hg(II) and PMA in excess of their available thiol ligands; X-ray absorption spectroscopy indicated nitrogens as likely additional ligands. The mercurials released protein-bound iron (Fe) more effectively than common organic oxidants and all disturbed the Na(+)/K(+) electrolyte balance, but none provoked efflux of six essential transition metals including Fe. PMA and MT made stable cysteine monothiol adducts in many Fe-binding proteins, but stable Hg(II) adducts were only seen in CysXxx(n)Cys peptides. We conclude that on acute exposure: (a) the distinct effects of mercurials on thiol and Fe homeostases reflected their different uptake and valences; (b) their similar effects on essential metal and electrolyte homeostases reflected the energy dependence of these processes; and (c) peptide phenylmercury-adducts were more stable or detectable in mass spectrometry than Hg(II)-adducts. These first in vivo observations in a well-defined model organism reveal differences upon acute exposure to inorganic and organic mercurials that may underlie their distinct toxicology.
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Escherichia coli/efectos de los fármacos , Homeostasis/efectos de los fármacos , Proteínas de Unión a Hierro/metabolismo , Mercurio/farmacología , Mercurio/toxicidad , Espectroscopía de Resonancia por Spin del Electrón , Contaminantes Ambientales/toxicidad , Compuestos Organomercuriales/toxicidad , Compuestos de SulfhidriloRESUMEN
The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.
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Oxidorreductasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Datos de Secuencia Molecular , Difracción de Neutrones , Oxidorreductasas/metabolismo , Unión Proteica , Dispersión del Ángulo Pequeño , Electricidad EstáticaRESUMEN
Mercuric reductase, MerA, is a key enzyme in bacterial mercury resistance. This homodimeric enzyme captures and reduces toxic Hg2+ to Hg0, which is relatively unreactive and can exit the cell passively. Prior to reduction, the Hg2+ is transferred from a pair of cysteines (C558' and C559' using Tn501 numbering) at the C-terminus of one monomer to another pair of cysteines (C136 and C141) in the catalytic site of the other monomer. Here, we present the X-ray structure of the C-terminal Hg2+ complex of the C136A/C141A double mutant of the Tn501 MerA catalytic core and explore the molecular mechanism of this Hg transfer with quantum mechanical/molecular mechanical (QM/MM) calculations. The transfer is found to be nearly thermoneutral and to pass through a stable tricoordinated intermediate that is marginally less stable than the two end states. For the overall process, Hg2+ is always paired with at least two thiolates and thus is present at both the C-terminal and catalytic binding sites as a neutral complex. Prior to Hg2+ transfer, C141 is negatively charged. As Hg2+ is transferred into the catalytic site, a proton is transferred from C136 to C559' while C558' becomes negatively charged, resulting in the net transfer of a negative charge over a distance of â¼7.5 Å. Thus, the transport of this soft divalent cation is made energetically feasible by pairing a competition between multiple Cys thiols and/or thiolates for Hg2+ with a competition between the Hg2+ and protons for the thiolates.
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Proteínas Bacterianas/química , Mercurio/metabolismo , Oxidorreductasas/química , Pseudomonas aeruginosa/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/química , Cisteína/metabolismo , Modelos Moleculares , Oxidorreductasas/metabolismo , Conformación Proteica , Multimerización de Proteína , Pseudomonas aeruginosa/metabolismoRESUMEN
The South African lion (Panthera leo) population is highly fragmented. One-third of its wild lions occur in small (<1000 km(2)) reserves. These lions were reintroduced from other areas of the species' historical range. Management practices on these reserves have not prioritized genetic provenance or heterozygosity. These trends potentially constrain the conservation value of these lions. To ensure the best management and long-term survival of these subpopulations as a viable collective population, the provenance and current genetic diversity must be described. Concurrently, poaching of lions to supply a growing market for lion bones in Asia may become a serious conservation challenge in the future. Having a standardized, validated method for matching confiscated lion parts with carcasses will be a key tool in investigating these crimes. We evaluated 28 microsatellites in the African lion using samples from 18 small reserves and 1 captive facility in South Africa, two conservancies in Zimbabwe, and Kruger National and Kgalagadi Transfrontier Parks to determine the loci most suited for population management and forensic genetic applications. Twelve microsatellite loci with a match probability of 1.1×10(-5) between siblings were identified for forensics. A further 10 could be added for population genetics studies.
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Conservación de los Recursos Naturales/métodos , Genética de Población/métodos , Leones/genética , Repeticiones de Microsatélite , Alelos , Animales , ADN/aislamiento & purificación , Femenino , Genética Forense , Masculino , Análisis de Secuencia de ADN , SudáfricaRESUMEN
Health care workers experience high rates of burnout and psychiatric distress. A large health care system in the southwest United States developed a comprehensive mental health service model for employees. Services offered range from traditional benefits (eg, Employee Assistance Program), resiliency and well-being initiatives, and innovative technology solutions, to access to peer support services for professional practice issues. The latest innovation in services is a free, self-insured outpatient mental health clinic designed exclusively for health care workers and their dependents. In this article, the authors describe the development of expanded mental health programming for health care workers and discuss how this unique service model proactively reduces common barriers to the receipt of high-quality care. This approach to caring for the workforce may serve as a model for other health care organizations across the United States. By providing mental health support to employees, health care organizations are mitigating the risk of burnout and related consequences to the system.
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Agotamiento Profesional , Personal de Salud , Servicios de Salud Mental , Humanos , Agotamiento Profesional/prevención & control , Agotamiento Profesional/psicología , Personal de Salud/psicología , Sudoeste de Estados Unidos , Estados Unidos , AdultoRESUMEN
The identification of peptides that result from post-translational modifications is critical for understanding normal pathways of cellular regulation as well as identifying damage from, or exposures to xenobiotics, i.e. the exposome. However, because of their low abundance in proteomes, effective detection of modified peptides by mass spectrometry (MS) typically requires enrichment to eliminate false identifications. We present a new method for confidently identifying peptides with mercury (Hg)-containing adducts that is based on the influence of mercury's seven stable isotopes on peptide isotope distributions detected by high-resolution MS. Using a pure protein and E. coli cultures exposed to phenyl mercuric acetate, we show the pattern of peak heights in isotope distributions from primary MS single scans efficiently identified Hg adducts in data from chromatographic separation coupled with tandem mass spectrometry with sensitivity and specificity greater than 90%. Isotope distributions are independent of peptide identifications based on peptide fragmentation (e.g. by SEQUEST), so both methods can be combined to eliminate false positives. Summing peptide isotope distributions across multiple scans improved specificity to 99.4% and sensitivity above 95%, affording identification of an unexpected Hg modification. We also illustrate the theoretical applicability of the method for detection of several less common elements including the essential element, selenium, as selenocysteine in peptides.
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Isótopos de Mercurio/química , Procesamiento Proteico-Postraduccional , Proteoma/química , Algoritmos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Escherichia coli , Proteínas de Escherichia coli/química , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Marcaje Isotópico , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteómica , Conejos , Relación Señal-Ruido , Espectrometría de Masas en TándemRESUMEN
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, we demonstrate complex formation between TclI, TclJ and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core.
RESUMEN
INTRODUCTION: The objectives of this study were to develop and evaluate a curriculum that integrated biostatistics and research design content with core sciences content within a pharmacy course. METHODS: An inquiry curriculum was developed in 2019 and included lectures on biostatistics and research design with small group discussions of clinical research papers directly related to the core sciences content. Students' perceptions and pass rates between students who did (2019 cohort) and did not (2018 cohort) undergo the curriculum were compared. Test scores taken approximately one year after completion of each cohort's course were also compared. RESULTS: Of 127 students in the 2019 cohort, 120 (94%) responded. Over 90% agreed or strongly agreed that inquiry and core sciences contents were integrated well. The 2019 cohort had a significantly higher pass rate than the 2018 cohort on two of three assessment questions evaluated: one multiple choice question (P = .037) and one short answer question (P = .013). After adjustments for baseline characteristics, retention study volunteers from the 2019 cohort had a significantly higher percent test score than those from the 2018 cohort (parameter estimate = 8.48%; P = .026). CONCLUSIONS: An inquiry curriculum consisting of select biostatistics and research design topics can be integrated with a core sciences curriculum in a large integrated pharmacy course. Inclusion of this content increased student academic performance and retention of knowledge and skills.
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Bioestadística , Evaluación Educacional , Estudios de Cohortes , Curriculum , Humanos , Proyectos de InvestigaciónRESUMEN
Conservation translocations have become an important management tool, particularly for large wildlife species such as the lion (Panthera leo). When planning translocations, the genetic background of populations needs to be taken into account; failure to do so risks disrupting existing patterns of genetic variation, ultimately leading to genetic homogenization, and thereby reducing resilience and adaptability of the species. We urge wildlife managers to include knowledge of the genetic background of source/target populations, as well as species-wide patterns, in any management intervention. We present a hierarchical decision-making tool in which we list 132 lion populations/lion conservation units and provide information on genetic assignment, uncertainty and suitability for translocation for each source/target combination. By including four levels of suitability, from 'first choice' to 'no option', we provide managers with a range of options. To illustrate the extent of international trade of lions, and the potential disruption of natural patterns of intraspecific diversity, we mined the CITES Trade Database for estimated trade quantities of live individuals imported into lion range states during the past 4 decades. We identified 1056 recorded individuals with a potential risk of interbreeding with wild lions, 772 being captive-sourced. Scoring each of the records with our decision-making tool illustrates that only 7% of the translocated individuals were 'first choice' and 73% were 'no option'. We acknowledge that other, nongenetic factors are important in the decision-making process, and hence a pragmatic approach is needed. A framework in which source/target populations are scored based on suitability is not only relevant to lion, but also to other species of wildlife that are frequently translocated. We hope that the presented overview supports managers to include genetics in future management decisions and contributes towards conservation of the lion in its full diversity.
RESUMEN
Critical care professionals in the United States are experiencing distress and frustration during the recent delta-wave of the coronavirus disease 2019 pandemic. This wave feels different because most, although not all, patients suffering with the sequelae from coronavirus disease 2019 who enter ICUs are unvaccinated. Since vaccines in the United States are safe, effective, and widely available for people 12 and older, severe cases of coronavirus disease 2019 are now considered preventable. However, even when a disease is preventable, critical care professionals still have remaining role-based, ethical obligations to their patients. Developing additional mechanisms for reflection and resilience, in spite of accumulated frustrations from otherwise preventable mortality, may help the professional and those they care for. In this essay, we propose a number of questions that recognize the existential frustrations critical care professionals experience, while also uncovering the ethical obligations that remain. Rather than becoming comfortable with silence or frustration, these reflections intend to bridge the gap between feeling frustrated and building relationships that benefit both the patient and the critical care professional during this pandemic.
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To avoid nonspecific and/or undesirable binding and reactivity of metal ions with cellular components, organisms have evolved metal-specific systems for trafficking proteins. Although systems differ, those handling soft metal ions such as Hg(2+), Cu(+), Zn(2+), etc., all utilize heavy metal-associated (HMA) proteins and domains of ~70 amino acids with a conserved GMXCXXC motif in a ßαßßαß structural fold. While the conserved cysteines define a common metal binding site in these proteins, other structural features must be utilized to create metal ion, protein partner, and contextual specificities. This paper presents initial structure-function studies of the N-terminal HMA domain (NmerA) of Tn501 mercuric ion reductase (MerA) aimed at identifying structural features critical to its role in facilitating efficient transfer of Hg(2+) to the MerA catalytic core for reductive detoxification. First, NMR solution structures of reduced and Hg(2+)-bound forms of NmerA are presented that allow definition and comparison of the structure of the metal binding loop in the two states. Structural differences between the two forms are compared with differences observed in three HMA domains with different metal ion and functional contexts. Second, analyses of the UV absorbance properties of wild-type, Cys11Ala, and Cys14Ala forms of NmerA are presented that provide assignments of the pK(a) values for the two cysteine thiols of the metal binding motif. Third, results from ¹³C NMR studies with wild-type and Y62F NmerA labeled with [ß-¹³C]cysteine are presented that define a role for Tyr62 in modulating the pK(a) values of the cysteine thiols.
Asunto(s)
Bacterias/enzimología , Cisteína/química , Elementos Transponibles de ADN , Metales Pesados/química , Oxidorreductasas/química , Secuencias de Aminoácidos , Cisteína/metabolismo , Cinética , Metales Pesados/metabolismo , Oxidorreductasas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Aerobic and facultative bacteria and archaea harboring mer loci exhibit resistance to the toxic effects of Hg(II) and organomercurials [RHg(I)]. In broad spectrum resistance, RHg(I) is converted to less toxic Hg(0) in the cytosol by the sequential action of organomercurial lyase (MerB: RHg(I) â RH + Hg(II)) and mercuric ion reductase (MerA: Hg(II) â Hg(0)) enzymes, requiring transfer of Hg(II) from MerB to MerA. Although previous studies with γ-proteobacterial versions of MerA and a nonphysiological Hg(II)-DTT-MerB complex qualitatively support a pathway for direct transfer between proteins, assessment of the relative efficiencies of Hg(II) transfer to the two different dicysteine motifs in γ-proteobacterial MerA and to competing cellular thiol is lacking. Here we show the intrinsic tryptophan fluorescence of γ-proteobacterial MerB is sensitive to Hg(II) binding and use this to probe the kinetics of Hg(II) removal from MerB by the N-terminal domain (NmerA) and catalytic core C-terminal cysteine pairs of its coevolved MerA and by glutathione (GSH), the major competing cellular thiol in γ-proteobacteria. At physiologically relevant concentrations, reaction with a 10-fold excess of NmerA over HgMerB removes ≥92% Hg(II), while similar extents of reaction require more than 1000-fold excess of GSH. Kinetically, the apparent second-order rate constant for Hg(II) transfer from MerB to NmerA, at (2.3 ± 0.1) × 10(4) M(-1) s(-1), is â¼100-fold greater than that for GSH ((1.2 ± 0.2) × 10(2) M(-1) s(-1)) or the MerA catalytic core (1.2 × 10(2) M(-1) s(-1)), establishing transfer to the metallochaperone-like NmerA domain as the kinetically favored pathway in this coevolved system.
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Liasas/metabolismo , Mercurio/metabolismo , Oxidorreductasas/metabolismo , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Dominio Catalítico , Cisteína/química , Cisteína/metabolismo , Fluorescencia , Gammaproteobacteria/metabolismo , Glutatión/farmacología , Iones/farmacología , Cinética , Mercurio/química , Mercurio/farmacología , Metalochaperonas , Datos de Secuencia Molecular , Triptófano/farmacologíaRESUMEN
Demethylation is a key reaction in global mercury cycling. The bacterial organomercurial lyase, MerB, catalyzes the demethylation of a wide range of organomercurials via Hg-C protonolysis. Two strictly conserved cysteine residues in the active site are required for catalysis, but the source of the catalytic proton and the detailed reaction mechanism have not been determined. Here, the two major proposed reaction mechanisms of MerB are investigated and compared using hybrid density functional theory calculations. A model of the active site was constructed from an X-ray crystal structure of the Hg(II)-bound MerB product complex. Stationary point structures and energies characterized for the Hg-C protonolysis of methylmercury rule out the direct protonation mechanism in which a cysteine residue delivers the catalytic proton directly to the organic leaving group. Instead, the calculations support a two-step mechanism in which Cys96 or Cys159 first donates a proton to Asp99, enabling coordination of two thiolates with R-Hg(II). At the rate-limiting transition state, Asp99 protonates the nascent carbanion in a trigonal planar, bis thiol-ligated R-Hg(II) species to cleave the Hg-C bond and release the hydrocarbon product. Reactions with two other substrates, vinylmercury and cis-2-butenyl-2-mercury, were also modeled, and the computed activation barriers for all three organomercurial substrates reproduce the trend in the experimentally observed enzymatic reaction rates. Analysis of atomic charges in the rate-limiting transition state structure using Natural Population Analysis shows that MerB lowers the activation free energy in the Hg-C protonolysis reaction by redistributing electron density into the leaving group and away from the catalytic proton.
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Carbono/metabolismo , Liasas/metabolismo , Mercurio/metabolismo , Modelos Moleculares , Biocatálisis , Dominio Catalítico , Liasas/química , Teoría Cuántica , Agua/metabolismoRESUMEN
PURPOSE: The factorial and concurrent validity of the Moorong Self-Efficacy Scale (MSES) was examined using a sample of Americans with spinal cord injury. METHODS: One hundred sixty-two participants were recruited with the assistance of the Florida Brain and Spinal Cord Injury Program and the Florida Spinal Cord Injury Resource Centre. Mean age of participants was 45.8 years (SD = 13.4), and 68.5% were men. The participants completed a survey containing a demographic questionnaire, the MSES, the Satisfaction with Life Scale (SWLS), the Personal Resources Questionnaire--2000 (PRQ-2000), and the Centre for Epidemiologic Studies Depression Scale--10 item version (CES-D-10). RESULTS: Factor analysis yielded two factors (Interpersonal Self-Efficacy and Instrumental Self-Efficacy) similar to the original MSES. In addition, the MSES factors and total score in the present study were significantly correlated in the predicted directions with specified psychosocial variables, as well as hours of paid employment. CONCLUSIONS: The results of this study support the factorial and concurrent validity of the MSES as a self-efficacy measure in Americans with spinal cord injury.