RESUMO
Pathogenic bacteria, including drug-resistant variants such as methicillin-resistant Staphylococcus aureus (MRSA), can cause severe infections in the human body. Early detection of MRSA is essential for clinical diagnosis and proper treatment, considering the distinct therapeutic strategies for methicillin-sensitive S. aureus (MSSA) and MRSA infections. However, the similarities between MRSA and MSSA properties present a challenge in promptly and accurately distinguishing between them. This work introduces an approach to differentiate MRSA from MSSA utilizing matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in conjunction with a neural network-based classification model. Four distinct strains of S. aureus were utilized, comprising three MSSA strains and one MRSA strain. The classification accuracy of our model ranges from ~ 92 to ~ 97% for each strain. We used deep SHapley Additive exPlanations to reveal the unique feature peaks for each bacterial strain. Furthermore, Fe3O4 MNPs were used as affinity probes for sample enrichment to eliminate the overnight culture and reduce the time in sample preparation. The limit of detection of the MNP-based affinity approach toward S. aureus combined with our machine learning strategy was as low as ~ 8 × 103 CFU mL-1. The feasibility of using the current approach for the identification of S. aureus in juice samples was also demonstrated.
Assuntos
Nanopartículas de Magnetita , Staphylococcus aureus Resistente à Meticilina , Humanos , Staphylococcus aureus , Meticilina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Aprendizado de MáquinaRESUMO
Autonomous locomotion is a ubiquitous phenomenon in biology and in physics of active systems at microscopic scale. This includes prokaryotic, eukaryotic cells (crawling and swimming) and artificial swimmers. An outstanding feature is the ability of these entities to follow complex trajectories, ranging from straight, curved (circular, helical...), to random-like ones. The non-straight nature of these trajectories is often explained as a consequence of the asymmetry of the particle or the medium in which it moves, or due to the presence of bounding walls, etc... Here, we show that for a particle driven by a concentration field of an active species, straight, circular and helical trajectories emerge naturally in the absence of asymmetry of the particle or that of suspending medium. Our proof is based on general considerations, without referring to an explicit form of a model. We show that these three trajectories correspond to self-congruent solutions. Self-congruency means that the states of the system at different moments of time can be made identical by an appropriate combination of rotation and translation of the coordinate space. We show that these solutions are exhibited by spherically symmetric particles as a result of a series of pitchfork bifurcations, leading to spontaneous symmetry breaking in the concentration field driving the particle motility. Self-congruent dynamics in one and two dimensions are analyzed as well. Finally, we present a simple explicit nonlinear exactly solvable model of fully isotropic phoretic particle that shows the transitions from a non-motile state to straight motion to circular motion to helical motion as a series of spontaneous symmetry-breaking bifurcations. Whether a system exhibits or not a given trajectory only depends on the numerical values of parameters entering the model, while asymmetry of swimmer shape, or anisotropy of the suspending medium, or influence of bounding walls are not necessary.
RESUMO
Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.
Assuntos
Aspergillus nidulans/metabolismo , Compostos de Epóxi/metabolismo , Proteínas Fúngicas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigênio/metabolismo , Oxigenases/metabolismo , Aspergillus nidulans/química , Aspergillus nidulans/enzimologia , Cristalografia por Raios X , Compostos de Epóxi/química , Proteínas Fúngicas/química , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Oxigênio/química , Oxigenases/químicaRESUMO
The swimming of a rigid phoretic particle in an isotropic fluid is studied numerically as a function of the dimensionless solute emission rate (or Péclet number Pe). The particle sets into motion at a critical Pe. Whereas the particle trajectory is straight at a small enough Pe, it is found that it loses its stability at a critical Pe in favor of a meandering motion. When Pe is increased further, the particle meanders at a short scale but its trajectory wraps into a circle at a larger scale. Increasing even further, Pe causes the swimmer to escape momentarily the circular trajectory in favor of chaotic motion, which lasts for a certain time, before regaining a circular trajectory, and so on. The chaotic bursts become more and more frequent as Pe increases, until the trajectory becomes fully chaotic, via the intermittency scenario. The statistics of the trajectory is found to be of the run-and-tumble-like nature at a short enough time and of diffusive nature at a long time without any source of noise.
RESUMO
Human type II topoisomerase (Top2) isoforms, hTop2α and hTop2ß, are targeted by some of the most successful anticancer drugs. These drugs induce Top2-mediated DNA cleavage to trigger cell-death pathways. The potency of these drugs correlates positively with their efficacy in stabilizing the enzyme-mediated DNA breaks. Structural analysis of hTop2α and hTop2ß revealed the presence of methionine residues in the drug-binding pocket, we therefore tested whether a tighter Top2-drug association may be accomplished by introducing a methionine-reactive Pt2+ into a drug to further stabilize the DNA break. Herein, we synthesized an organoplatinum compound, etoplatin-N2ß, by replacing the methionine-juxtaposing group of the drug etoposide with a cis-dichlorodiammineplatinum(II) moiety. Compared to etoposide, etoplatin-N2ß more potently inhibits both human Top2s. While the DNA breaks arrested by etoposide can be rejoined, those captured by etoplatin-N2ß are practically irreversible. Crystallographic analyses of hTop2ß complexed with DNA and etoplatin-N2ß demonstrate coordinate bond formation between Pt2+ and a flanking methionine. Notably, this stable coordinate tether can be loosened by disrupting the structural integrity of drug-binding pocket, suggesting that Pt2+ coordination chemistry may allow for the development of potent inhibitors with protein conformation-dependent reversibility. This approach may be exploited to achieve isoform-specific targeting of human Top2s.
Assuntos
Antineoplásicos/química , Quebras de DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Compostos Organoplatínicos/química , Podofilotoxina/análogos & derivados , Inibidores da Topoisomerase II/química , Antígenos de Neoplasias/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA/química , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Células HL-60 , Humanos , Metionina/química , Compostos Organoplatínicos/farmacologia , Podofilotoxina/química , Podofilotoxina/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose , Conformação Proteica , Inibidores da Topoisomerase II/farmacologiaRESUMO
Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.
Assuntos
Proteínas de Transporte/química , Homeostase , Ornitina Descarboxilase/química , Poliaminas/química , Proteínas/química , Sequência de Aminoácidos , Biocatálise , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteólise , Homologia de Sequência de AminoácidosRESUMO
AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3â epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived FeIV -oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.
Assuntos
Compostos de Epóxi/metabolismo , Proteínas Fúngicas/metabolismo , Peptídeos/metabolismo , Aspergillus nidulans/enzimologia , Biocatálise , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/metabolismo , Compostos de Epóxi/química , Compostos Férricos/química , Proteínas Fúngicas/química , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Simulação de Dinâmica Molecular , Peptídeos/química , Teoria Quântica , EstereoisomerismoRESUMO
A mesoscopic continuum model is employed to analyze the transport mechanisms and structure formation during the redistribution stage of deposition experiments where organic molecules are deposited on a solid substrate with periodic stripe-like wettability patterns. Transversally invariant ridges located on the more wettable stripes are identified as very important transient states and their linear stability is analyzed accompanied by direct numerical simulations of the fully nonlinear evolution equation for two-dimensional substrates. It is found that there exist two different instability modes that lead to different nonlinear evolutions that result (i) at large ridge volume in the formation of bulges that spill from the more wettable stripes onto the less wettable bare substrate and (ii) at small ridge volume in the formation of small droplets located on the more wettable stripes. In addition, the influence of different transport mechanisms during redistribution is investigated focusing on the cases of convective transport with no-slip at the substrate, transport via diffusion in the film bulk and via diffusion at the film surface. In particular, it is shown that the transport process does neither influence the linear stability thresholds nor the sequence of morphologies observed in the time simulation, but only the ratio of the time scales of the different process phases.
Assuntos
Compostos Orgânicos/química , Difusão , Tamanho da Partícula , Propriedades de Superfície , MolhabilidadeRESUMO
Peptidoglycan (PG) sacculi surround the cytoplasmic membrane, maintaining cell integrity by withstanding internal turgor pressure. During cell growth, PG endopeptidases cleave the crosslinks of the fully closed sacculi, allowing for the incorporation of new glycan strands and expansion of the peptidoglycan mesh. Outer-membrane-anchored NlpI associates with hydrolases and synthases near PG synthesis complexes, facilitating spatially close PG hydrolysis. Here, we present the structure of adaptor NlpI in complex with the endopeptidase MepS, revealing atomic details of how NlpI recruits multiple MepS molecules and subsequently influences PG expansion. NlpI binding elicits a disorder-to-order transition in the intrinsically disordered N-terminal of MepS, concomitantly promoting the dimerization of monomeric MepS. This results in the alignment of two asymmetric MepS dimers respectively located on the two opposite sides of the dimerization interface of NlpI, thus enhancing MepS activity in PG hydrolysis. Notably, the protein level of MepS is primarily modulated by the tail-specific protease Prc, which is known to interact with NlpI. The structure of the Prc-NlpI-MepS complex demonstrates that NlpI brings together MepS and Prc, leading to the efficient MepS degradation by Prc. Collectively, our results provide structural insights into the NlpI-enabled avidity effect of cellular endopeptidases and NlpI-directed MepS degradation by Prc.
Assuntos
Endopeptidases , Lipoproteínas , Peptidoglicano , Peptidoglicano/metabolismo , Endopeptidases/metabolismo , Endopeptidases/química , Lipoproteínas/metabolismo , Lipoproteínas/química , Ligação Proteica , Multimerização Proteica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Modelos Moleculares , Cristalografia por Raios X , Hidrólise , Escherichia coli/metabolismoRESUMO
Coffee is a daily essential, with prices varying based on taste, aroma, and chemical composition. However, distinguishing between different coffee beans is challenging due to time-consuming and destructive sample pretreatment. This study presents a novel approach for directly analyzing single coffee beans through mass spectrometry (MS) without the need for sample pretreatment. Using a single coffee bean deposited with a solvent droplet containing methanol and deionized water, we generated electrospray to extract the main species for MS analysis. Mass spectra of single coffee beans were obtained in just a few seconds. To showcase the effectiveness of the developed method, we used palm civet coffee beans (kopi luwak), one of the most expensive coffee types, as model samples. Our approach distinguished palm civet coffee beans from regular ones with high accuracy, sensitivity, and selectivity. Moreover, we employed a machine learning strategy to rapidly classify coffee beans based on their mass spectra, achieving 99.58% accuracy, 98.75% sensitivity, and 100% selectivity in cross-validation. Our study highlights the potential of combining the single-bean MS method with machine learning for the rapid and non-destructive classification of coffee beans. This approach can help to detect low-priced coffee beans mixed with high-priced ones, benefiting both consumers and the coffee industry.
Assuntos
Coffea , Animais , Coffea/química , Viverridae , Sementes/química , Espectrometria de Massas , Análise EspectralRESUMO
DNA gyrase is the only topoisomerase capable of introducing (-) supercoils into relaxed DNA. The C-terminal domain of the gyrase A subunit (GyrA-CTD) and the presence of a gyrase-specific 'GyrA-box' motif within this domain are essential for this unique (-) supercoiling activity by allowing gyrase to wrap DNA around itself. Here we report the crystal structure of Xanthomonas campestris GyrA-CTD and provide the first view of a canonical GyrA-box motif. This structure resembles the GyrA-box-disordered Escherichia coli GyrA-CTD, both adopting a non-planar beta-pinwheel fold composed of six seemingly spirally arranged beta-sheet blades. Interestingly, structural analysis revealed that the non-planar architecture mainly stems from the tilted packing seen between blades 1 and 2, with the packing geometry likely being defined by a conserved and unusual beta-strand-bearing proline. Consequently, the GyrA-box-containing blade 1 is placed at an angled spatial position relative to the other DNA-binding blades, and an abrupt bend is introduced into the otherwise flat DNA-binding surface. Mutagenesis studies support that the proline-induced structural twist contributes directly to gyrase's (-) supercoiling activity. To our knowledge, this is the first demonstration that a beta-strand-bearing proline may impact protein function. Potential relevance of beta-strand-bearing proline to disease phenylketonuria is also noted.
Assuntos
DNA Girase/química , Proteínas de Ligação a DNA/química , Prolina/química , Xanthomonas campestris/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , DNA Girase/genética , DNA Girase/metabolismo , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Prolina/análise , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways' end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. Here, we report the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT), which represents the first structural elucidation of the GSMT methyltransferase family. As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. This work implicates a structural basis by which feedback inhibition of biosynthetic methyltransferases may be achieved.
Assuntos
Glicina N-Metiltransferase/química , Glicina N-Metiltransferase/metabolismo , Methanosarcinaceae/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Betaína/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Retroalimentação Fisiológica , Regulação da Expressão Gênica em Archaea , Regulação Enzimológica da Expressão Gênica , Glicina/metabolismo , Methanosarcinaceae/química , Metilação , Modelos Moleculares , Estrutura Secundária de Proteína , Sarcosina/metabolismoRESUMO
Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2ß complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions provides information applicable for developing an isoform-specific TOP2-targeting strategy.