RESUMO
Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.
Assuntos
Aminoglicosídeos/química , Antibacterianos/química , Oxigênio/química , Oxigenases/química , Domínio Catalítico , Cristalografia por Raios X , Ciclização , Hidrogênio/química , Higromicina B/química , Metais/química , Micromonospora/enzimologia , Micromonospora/genética , Família Multigênica , Oligossacarídeos/química , Fases de Leitura Aberta , Oxirredução , Filogenia , Ligação Proteica , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina Difosfato/química , Regulação Alostérica/fisiologia , Substituição de Aminoácidos , Animais , Cristalografia por Raios X , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , RatosRESUMO
Respiratory processes often use quinone oxidoreduction to generate a transmembrane proton gradient, making the 2H(+)/2e(-) quinone chemistry important for ATP synthesis. There are a variety of quinones used as electron carriers between bioenergetic proteins, and some respiratory proteins can functionally interact with more than one quinone type. In the case of complex II homologs, which couple quinone chemistry to the interconversion of succinate and fumarate, the redox potentials of the biologically available ubiquinone and menaquinone aid in driving the chemical reaction in one direction. In the complex II homolog quinol:fumarate reductase, it has been demonstrated that menaquinol oxidation requires at least one proton shuttle, but many of the remaining mechanistic details of menaquinol oxidation are not fully understood, and little is known about ubiquinone reduction. In the current study, structural and computational studies suggest that the sequential removal of the two menaquinol protons may be accompanied by a rotation of the naphthoquinone ring to optimize the interaction with a second proton shuttling pathway. However, kinetic measurements of site-specific mutations of quinol:fumarate reductase variants show that ubiquinone reduction does not use the same pathway. Computational docking of ubiquinone followed by mutagenesis instead suggested redundant proton shuttles lining the ubiquinone-binding site or from direct transfer from solvent. These data show that the quinone-binding site provides an environment that allows multiple amino acid residues to participate in quinone oxidoreduction. This suggests that the quinone-binding site in complex II is inherently plastic and can robustly interact with different types of quinones.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Simulação de Acoplamento Molecular , Oxirredutases/química , Ubiquinona/química , Domínio Catalítico , Cinética , Relação Estrutura-AtividadeRESUMO
Coronary angiography has long been the standard for coronary imaging, but it has limitations in assessing vessel wall anatomy and guiding percutaneous coronary intervention (PCI). Intracoronary imaging techniques like intravascular ultrasound (IVUS) and optical coherence tomography (OCT) can overcome these limitations. IVUS uses ultrasound and OCT uses near-infrared light to visualize coronary pathology in unique ways due to differences in temporal and spatial resolution. These techniques have evolved to offer clinical utility in plaque characterization and vessel assessment during PCI. Meta-analyses and adjusted observational studies suggest that both IVUS and OCT-guided PCI correlate with reduced cardiovascular risks compared to angiographic guidance alone. While IVUS demonstrates consistent clinical outcome benefits, OCT evidence is less robust. IVUS has progressed from early motion detection to high-resolution systems, with smaller compatible catheters. OCT utilizes near infrared light to achieve unparalleled resolutions, but requires temporary blood clearance for optimal imaging. Enhanced visualization and guidance make IVUS and OCT well-suited for higher risk PCI in patients with diabetes and chronic kidney disease by allowing detailed visualization of complex lesions and ensuring optimal stent deployment and positioning in PCI for patients with type 2 diabetes and chronic kidney disease, improving outcomes. IVUS and recent advancements in zero- and low-contrast OCT techniques can reduce nephrotoxic contrast exposure, thus helping to minimize PCI complications in these high-risk patient groups. IVUS and OCT provide valuable insights into coronary pathophysiology and guide interventions precisely compared to angiography alone. Both have comparable clinical outcomes, emphasizing the need for tailored imaging choices based on clinical scenarios. Continued refinement and integration of intravascular imaging will likely play a pivotal role in optimizing coronary interventions and outcomes. This systematic review aims to delve into the nuances of IVUS and OCT, highlighting their strengths and limitations as PCI adjuncts.
RESUMO
INTRODUCTION: Peripheral arterial disease (PAD) is a progressive vascular condition characterized by the narrowing or blockage of arteries, primarily attributed to atherosclerosis. PAD's prevalence in the general population is estimated at approximately 5.9%. Notably however, among patients with chronic kidney disease (CKD), PAD's prevalence is substantially higher, ranging from 17% to 48%. This review paper emphasizes the pervasiveness of PAD and its intricate relationship with CKD and end-stage renal disease (ESRD). It demonstrates the importance of early detection, proactive screening, and understanding the formidable challenges associated with treating heavily calcified lesions. EVIDENCE ACQUISITION: Comprehensive literature searches encompassed the PubMed/MEDLINE, Cochrane Library, and Embase databases, in order to identify studies involving lower extremity peripheral arterial interventions in patients both with and without CKD or ESRD. The search spanned the timeframe from January 2001 to July 2023. The search strategy included vocabulary terms concerning peripheral artery disease, lower extremities, revascularization, chronic kidney disease, and end-stage renal disease. EVIDENCE SYNTHESIS: Initial searches were used to identify articles based on title. Exclusion criteria was then applied, and any redundant articles were removed. The articles abstracts were then reviewed, and relevant articles were selected. Once selected the articles were thoroughly reviewed including the references to find other relevant articles that were missed during the initial search process. In total 28 articles were selected and included for review of clinical data in regard to PAD outcomes in patients with advanced kidney disease. CONCLUSIONS: The findings highlight the need for personalized approaches in diagnosing and treating PAD in CKD and ESRD patients. Interdisciplinary collaboration, such as those between nephrologists, vascular surgeons, and interventional radiologists, is vital to optimize outcomes. Further research should focus on innovative, tailored interventions to enhance limb preservation, reduce mortality, prolong patency, and cut healthcare costs.
Assuntos
Falência Renal Crônica , Doença Arterial Periférica , Insuficiência Renal Crônica , Humanos , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/epidemiologia , Falência Renal Crônica/complicações , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/terapia , Extremidade Inferior , Rim , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/terapiaRESUMO
Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.