RESUMEN
Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.
Asunto(s)
Streptomyces , Vanadio , Antibacterianos/química , Catálisis , Isomerasas , Vanadio/químicaRESUMEN
Von Willebrand Factor (VWF) has a central role in primary hemostasis. Its biological activity is related to the size of VWF multimers, spontaneously binding to platelets and inducing circulating microthrombi formation. This process is down-regulated by the VWF cleaving protease ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin motif). To date, information regarding the levels of ADAMTS13 in neonates and preterm infants is scarce. Our aim was to study ADAMTS13, VWF antigen (Ag) and Ristocetin cofactor (RiCof) activity in neonates and evaluate potential correlations with perinatal complications. Our cohort consisted of 128 (48/128: born preterm) neonates, born in Sheba Medical Center and followed until hospital discharge. Control group consisted of 20 healthy adults. As expected, a significant elevation of VWF:Ag was observed in preterm and term infants compared to adults. VWF:Ag levels were highest in full term infants (Median 129.0 IQR 33.8) and lowest in adults (Median 119.0 IQR 58.5) (p<0.05), and RiCoF levels in neonates were higher than in adults. ADAMTS13 was significantly (p<0.05) higher in preterm babies in comparison to full term and adult controls. Neonates that underwent stressful conditions or experienced vascular complications such as IUGR, ROP, NEC, had lower levels of ADAMTS13 in our study. Further studies are required to validate and asses potential significance of these findings.
Asunto(s)
Proteína ADAMTS13/sangre , Recien Nacido Prematuro/sangre , Nacimiento Prematuro/sangre , Factor de von Willebrand/análisis , Proteína ADAMTS13/metabolismo , Adulto , Estudios de Cohortes , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro/metabolismo , Persona de Mediana Edad , Embarazo , Nacimiento Prematuro/metabolismo , Adulto Joven , Factor de von Willebrand/metabolismoRESUMEN
Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.
Asunto(s)
Dioxigenasas/química , Dioxigenasas/metabolismo , Oxígeno/metabolismo , Streptomyces/enzimología , Vancomicina/biosíntesis , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión/genética , Catálisis , Coenzima A/metabolismo , Dioxigenasas/genética , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Químicos , Modelos Moleculares , Mutación/genética , Conformación Proteica , Streptomyces/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Vancomicina/química , Vancomicina/metabolismoRESUMEN
The pyrroloquinoline alkaloid family of natural products, which includes the immunosuppressant lymphostin, has long been postulated to arise from tryptophan. We now report the molecular basis of lymphostin biosynthesis in three marine Salinispora species that maintain conserved biosynthetic gene clusters harboring a hybrid nonribosomal peptide synthetase-polyketide synthase that is central to lymphostin assembly. Through a series of experiments involving gene mutations, stable isotope profiling, and natural product discovery, we report the assembly-line biosynthesis of lymphostin and nine new analogues that exhibit potent mTOR inhibitory activity.
Asunto(s)
Actinomycetales/metabolismo , Alcaloides/metabolismo , Alcaloides/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Pirroles/metabolismo , Pirroles/farmacología , Quinolinas/metabolismo , Quinolinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Actinomycetales/química , Alcaloides/química , Inhibidores Enzimáticos/química , Pirroles/química , Quinolinas/química , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Prephenate is the direct precursor of phenylpyruvate and 4-hydroxyphenylpyruvate in the biogenesis of phenylalanine and tyrosine by action of the decarboxylative, aromatizing enzymes prephenate dehydratase and dehydrogenase, respectively. The recent characterization of BacA in bacilysin biosynthesis as a nonaromatizing decarboxylase reveals a new route from prephenate in the biosynthesis of nonproteinogenic amino acids. This study describes two additional enzymes, AerD from Planktothrix agardhii and SalX from Salinispora tropica, that utilize the central building block prephenate for flux down distinct pathways to amino acid products, representing a new metabolic fate for prephenate and establishing a new family of nonaromatizing prephenate decarboxylases.
Asunto(s)
Carboxiliasas/metabolismo , Ácidos Ciclohexanocarboxílicos/metabolismo , Ciclohexenos/metabolismo , Actinomycetales/enzimología , Actinomycetales/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carboxiliasas/genética , Cianobacterias/enzimología , Cianobacterias/genética , Ácidos Ciclohexanocarboxílicos/química , Ciclohexenos/química , Genes BacterianosRESUMEN
Without disregarding its role as one of the key sources of sustainable livelihoods in Zimbabwe and other developing countries, livestock production contributes significantly to greenhouse gas (GHG) emissions through enteric fermentation. For the livestock sector to complement global efforts to mitigate climate change, accurate estimations of GHG emissions are required. Methane emissions from enteric fermentation in Zimbabwe were quantified over 35years under four production systems and five agro-ecological regions. The Intergovernmental Panel on Climate Change emission factor methodology was used to derive CH4 emissions from seven livestock categories at national level. Emission intensities based on human population, domestic export of livestock meat and climate variables were used to assess emission drivers and predict future emission trends. Over the past 35years, enteric fermentation CH4 emissions from all livestock categories ranged between 158.3 and 204.3Ggyear-1. Communal lands, typified by indigenous livestock breeds, had the highest contribution of between 58% and 75% of the total annual emissions followed by livestock from large scale commercial (LSC) farms. The decreasing livestock population on LSC farms and consequent decline in production could explain the lack of a positive response of CH4 emissions to human population growth, and decreasing emissions per capita over time at -0.3kg CH4capita-1year-1. The emissions trend showed that even if Zimbabwe's national livestock population doubles in 2030 relative to the 2014 estimates, the country would still remain with similar magnitude of CH4 emission intensity as that of 1980. No significant correlations (P>0.05) were found between emissions and domestic export of beef and pork. Further research on enhanced characterisation of livestock species, population and production systems, as well as direct measurements and modelling of emissions from indigenous and exotic livestock breeds were recommended.
Asunto(s)
Contaminantes Atmosféricos/análisis , Monitoreo del Ambiente , Metano/análisis , Contaminación del Aire , Crianza de Animales Domésticos/métodos , Animales , Cambio Climático , Ganado , ZimbabweRESUMEN
The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.
Asunto(s)
Dioxigenasas/metabolismo , Oxígeno/metabolismo , Streptomyces/enzimología , Sitios de Unión , Cristalografía por Rayos X , Dioxigenasas/química , Dioxigenasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
BACKGROUND: Emerging adulthood is a challenging period for diabetes management. Our aim was to determine whether a dedicated transition clinic for emerging adults with type 1 diabetes can improve glycemic control and visit attendance. METHODS: An observational study of 53 emerging adults (30 males) treated during 2010-2014 in a newly established transition clinic. The clinic was operated jointly by pediatric and adult endocrinologists and included a transition coordinator. Data collected included the source of referral, HbA1c levels, frequency of visit attendance, and acute complications. For 27 patients who had attended the pediatric clinic at the same medical center, data from up to 2 years preceding the transition were also collected. Patients filled the Diabetes Quality of Life-Youth questionnaire at the transition and 1 year later. RESULTS: Mean ± SD age at the transfer to the transition clinic was 22.1 ± 2.7 years; mean disease duration was 8.4 ± 5.0 years. Follow-up duration at the transition clinic was 1.2 ± 1.1 years. Mean HbA1c levels decreased from 67 mmol/mol (95 % CI 63-72) [8.3 % (95 % CI 7.9-8.7)] at transfer to 57 mmol/mol (95 % CI 52-63) [7.4 % (95 % CI 6.9-7.9)] after 1 year (p < 0.001). Thirty-six patients (68 %) attended three or more visits during their first year in the transition clinic. The impact of diabetes on quality of life, disease-related worries, and life satisfaction did not change significantly during 1-year attendance in the transition clinic. CONCLUSIONS: A dedicated transition clinic for emerging adults, with tailored support according to the developmental needs of emerging adulthood, showed improved glycemic control and visit attendance.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/epidemiología , Participación del Paciente/estadística & datos numéricos , Cuidado de Transición/estadística & datos numéricos , Adolescente , Adulto , Atención Ambulatoria/estadística & datos numéricos , Glucemia/análisis , Niño , Preescolar , Diabetes Mellitus Tipo 1/terapia , Femenino , Humanos , Masculino , Calidad de Vida , Derivación y Consulta , Encuestas y Cuestionarios , Transición a la Atención de Adultos/estadística & datos numéricos , Adulto JovenRESUMEN
Shoes with rocker bottom soles are utilized by persons with diabetic peripheral neuropathy to reduce plantar pressures during gait. The risk of falls increases with age and is compounded by diabetic neuropathy. The purpose of this study was to analyze how rocker bottom shoes affect posture control of older adults (50-75 years old) and younger adults (20-35 years old) in response to posterior slide perturbations. The postural response to a posterior platform translation was normalized among subjects by applying the below threshold stepping velocity (BTSV) for each subject. The BTSV was the fastest velocity of platform translation that did not cause a stepping response while wearing the rocker bottom shoes. Joint excursion, time to first response, response time, and variability of mean peak joint angles were analyzed at the ankle, knee, hip, trunk, and head in the sagittal plane. The statistical analysis was a 2-factor mixed repeated measures design to determine interactions between and within shoe types and age groups. While wearing rocker bottom shoes, both age groups exhibited increased joint excursion, differences in time to initial response, and longer response time. The older group demonstrated decreased joint excursion and increased time to initial response compared to the younger group, as well as a significantly slower mean BTSV. These findings support the conclusion that in healthy older adults and in populations at risk for falls, the use of rocker bottom or other unstable shoes may increase the potential of falls when confronted with a standing perturbation such as a forceful slip or trip.
Asunto(s)
Accidentes por Caídas/prevención & control , Articulación del Tobillo/fisiología , Pie/fisiología , Marcha/fisiología , Zapatos , Adulto , Anciano , Fenómenos Biomecánicos , Diseño de Equipo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Presión , Valores de Referencia , Adulto JovenRESUMEN
The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC [Widboom, P. W., Fielding, E. N., Liu, Y., and Bruner, S. D. (2007) Nature 447, 342-345] confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.