RESUMEN
Cardiovascular diseases can be diagnosed with computer assistance when using the magnetic resonance imaging (MRI) image that is produced by the MRI sensor. Deep learning-based scribbling MRI image segmentation has demonstrated impressive results recently. However, the majority of current approaches possess an excessive number of model parameters and do not completely utilize scribbling annotations. To develop a feature decomposition distillation deep learning method, named FDDSeg, for scribble-supervised cardiac MRI image segmentation. Public ACDC and MSCMR cardiac MRI datasets were used to evaluate the segmentation performance of FDDSeg. FDDSeg adopts a scribble annotation reuse policy to help provide accurate boundaries, and the intermediate features are split class region and class-free region by using the pseudo labels to further improve feature learning. Effective distillation knowledge is then captured by feature decomposition. FDDSeg was compared with 7 state-of-the-art methods, MAAG, ShapePU, CycleMix, Dual-Branch, ZscribbleSeg, Perturbation Dual-Branch as well as ScribbleVC on both ACDC and MSCMR datasets. FDDSeg is shown to perform the best in DSC(89.05% and 88.75%), JC(80.30% and 79.78%) as well as HD95(5.76% and 4.44%) metrics with only 2.01M of parameters. FDDSeg methods can segment cardiac MRI images more precise with only scribble annotations at lower computation cost, which may help increase the efficiency of quantitative analysis of cardiac. Code and models are available at: https://github.com/labiip/FDDSeg.
RESUMEN
Engineering surfaces or membranes that allow an efficient oil/water separation is highly desired in a wide spectrum of applications ranging from oily wastewater discharge to offshore oil spill accidents. Recent advances in biomimetics, manufacturing, and characterization techniques have led to remarkable progress in the design of various superwetting materials with special wettability. In spite of exciting progress, formulating a strategy robust enough to guide the design and fabrication of separating surfaces remains a daunting challenge. In this review, we first present an overview of the wettability theory to elucidate how to control the surface morphology and chemistry to regulate oil/water separation. Then, parallel approaches are considered for discussing the separation mechanisms according to different oil/water mixtures, and three separation types were identified including filtration, adsorption and other separation types. Finally, perspectives on the challenges and future research directions in this research area are briefly discussed.
RESUMEN
Recently, deep neural network-based image compressed sensing methods have achieved impressive success in reconstruction quality. However, these methods (1) have limitations in sampling pattern and (2) usually have the disadvantage of high computational complexity. To this end, a fast multi-scale generative adversarial network (FMSGAN) is implemented in this paper. Specifically, (1) an effective multi-scale sampling structure is proposed. It contains four different kernels with varying sizes so that decompose, and sample images effectively, which is capable of capturing different levels of spatial features at multiple scales. (2) An efficient lightweight multi-scale residual structure for deep image reconstruction is proposed to balance receptive field size and computational complexity. The key idea is to apply smaller convolution kernel sizes in the multi-scale residual structure to reduce the number of operations while maintaining the receptive field. Meanwhile, the channel attention structure is employed for enriching useful information. Moreover, perceptual loss is combined with MSE loss and adversarial loss as the optimization function to recover a finer image. Numerous experiments show that our FMSGAN achieves state-of-the-art image reconstruction quality with low computational complexity.
RESUMEN
BACKGROUND: Tendon-ligament injuries (TLIs), including Achilles tendinopathy, cruciate ligament injury, tennis elbow, rotator cuff injury, patellar tendinopathy, and tibial tendinopathy, are common musculoskeletal soft injuries during physical activity. Matrix metalloproteinase-3 (MMP-3) gene polymorphisms have been implicated in the etiology of TLIs in several genetic association studies with inconsistent results. The purpose of this study was to collect and synthesize the current evidences on the association of MMP-3 polymorphisms and TLIs. METHODS: The search was conducted using PubMed, Web of Science, EMBASE, Cochrane Library, CNKI and Wanfang databases, prior to July, 2021. Newcastle Ottawa Scale was used to appraise the study quality. Strengths of association were represented by odds ratios (ORs) and 95% confidence intervals (95% CIs). RESULTS: Thirteen studies with 2871 cases and 4497 controls met the eligibility criteria, and each study was in high quality. The overall analyzes suggested rs3025058 was associated with an increased TLIs risk (5A vs. 6A, OR = 1.20, 95% CI 1.03-1.40, P = 0.020). However, the association was not found for rs679620, rs591058, and rs650108 polymorphisms. Subgroup analysis by injury type suggested that rs679620 polymorphism was associated with a reduced risk to Achilles tendon rupture (AA + AG vs. GG, OR = 0.46, 95% CI 0.25-0.87, P = 0.020), and rs3025058 was associated with an elevated risk to anterior cruciate ligament injury (5A5A + 5A6A vs. 6A6A, OR = 1.46, 95% CI 1.03-2.06, P = 0.030). When stratified by ethnicity, the findings indicated that rs3025058 polymorphism was associated with an increased TLIs risk among Caucasians (5A6A vs. 6A6A, OR = 1.55, 95% CI 1.09-2.42, P = 0.020) and Brazilians (5A5A vs. 5A6A + 6A6A, OR = 2.80, 95% CI 1.44-5.45, P = 0.002). CONCLUSION: Findings of this study suggest that rs679620 polymorphism is associated with a reduced Achilles tendon rupture risk, and rs3025058 polymorphism contributes to an increased TLIs risk in Caucasians and Brazilians. However, rs591058 and rs650108 polymorphisms do not show any association with TLIs.
RESUMEN
Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, a simple method to fabricate substrates patterned with micrometer-scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces is described. The wells can also concentrate and localize cells and beads for cell-based assays.
Asunto(s)
Rayos Láser , Luz , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (Tm) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher Tm compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.
RESUMEN
Maize often exhibits asynchronous pollination under abiotic and biotic stress conditions; however, the molecular basis of this developmental deficiency has not been elucidated. Tassel development is a key process affecting the anthesis-silking interval (ASI) in maize. In this study, we showed that pollen shedding was delayed and ASI was significantly increased in B73 and Chang7-2 inbred lines under water deficit conditions, which resulted in longer barren tip length and decreased yields under both controlled and field conditions. Comparative transcriptome analysis performed on immature tassels derived from plants grown under well-watered and water deficit conditions identified 1931 and 1713 differentially expressed genes (DEGs) in B73 and Chang7-2, respectively. Further, 28 differentially co-expressed transcription factors were identified across both lines. Collectively, we demonstrated that the molecular regulation of tassel development is associated with water deficit stress at early vegetative stage in maize. This finding extends our understanding of the molecular basis of maize tassel development during abiotic stress.
Asunto(s)
Deshidratación , Inflorescencia/crecimiento & desarrollo , Inflorescencia/metabolismo , Agua/metabolismo , Zea mays , Deshidratación/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes del Desarrollo , Genes de Plantas , Inflorescencia/genética , Polinización/genética , Sitios de Carácter Cuantitativo , Estaciones del Año , Transcriptoma/fisiología , Zea mays/genética , Zea mays/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might differentiate them from other NAL subgroup members. The individual specificities of NahE and PhdJ are governed by Asn-281/Glu-285 and Ser-278/Asp-282, respectively. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.
Asunto(s)
Aldehído-Liasas/química , Proteínas Bacterianas/química , Mycobacterium/enzimología , Oxo-Ácido-Liasas/química , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/metabolismo , Modelos Moleculares , Mycobacterium/química , Mycobacterium/metabolismo , Oxo-Ácido-Liasas/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Conformación Proteica , Multimerización de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
5-Halo-2-hydroxy-2,4-pentadienoates (5-halo-HPDs) are reportedly generated in the bacterial catabolism of halogenated aromatic hydrocarbons by the meta-fission pathway. The 5-halo-HPDs, where the halogen can be bromide, chloride, or fluoride, result in the irreversible inactivation of 4-oxalocrotonate tautomerase (4-OT), which precedes the enzyme that generates them. The loss of activity is due to the covalent modification of the nucleophilic amino-terminal proline. Mass spectral and crystallographic analysis of the modified enzymes indicates that inactivation of 4-OT by 5-chloro- and 5-bromo-2-hydroxy-2,4-pentadienoate follows a mechanism different from that for the inactivation of 4-OT by 5-fluoro-2-hydroxy-2,4-pentadienoate. The 5-chloro and 5-bromo derivatives undergo 4-OT-catalyzed tautomerization to their respective α,ß-unsaturated ketones followed by attack at C5 (by the prolyl nitrogen) with concomitant loss of the halide. For the 5-fluoro species, the presence of a small amount of the α,ß-unsaturated ketone could result in a Michael addition of the prolyl nitrogen to C4 followed by protonation at C3. The fluoride is not eliminated. These observations suggest that the inactivation of 4-OT by a downstream metabolite could hamper the efficacy of the pathway, which is the first time that such a bottleneck has been reported for the meta-fission pathway.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Isomerasas/metabolismo , Pseudomonas putida/enzimología , Cristalografía por Rayos X , Activación Enzimática , Ácidos Grasos Insaturados/química , Halogenación , Isomerasas/química , Cinética , Modelos Moleculares , Pseudomonas putida/química , Pseudomonas putida/metabolismoRESUMEN
Maize kernel development is a complex biological process that involves the temporal and spatial expression of many genes and fine gene regulation at a transcriptional and post-transcriptional level, and microRNAs (miRNAs) play vital roles during this process. To gain insight into miRNA-mediated regulation of maize kernel development, a deep-sequencing technique was used to investigate the dynamic expression of miRNAs in the embryo and endosperm at three developmental stages in B73. By miRNA transcriptomic analysis, we characterized 132 known miRNAs and six novel miRNAs in developing maize kernel, among which, 15 and 14 miRNAs were commonly differentially expressed between the embryo and endosperm at 9 days after pollination (DAP), 15 DAP and 20 DAP respectively. Conserved miRNA families such as miR159, miR160, miR166, miR390, miR319, miR528 and miR529 were highly expressed in developing embryos; miR164, miR171, miR393 and miR2118 were highly expressed in developing endosperm. Genes targeted by those highly expressed miRNAs were found to be largely related to a regulation category, including the transcription, macromolecule biosynthetic and metabolic process in the embryo as well as the vitamin biosynthetic and metabolic process in the endosperm. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that these miRNAs displayed a negative correlation with the levels of their corresponding target genes. Importantly, our findings revealed that members of the miR169 family were highly and dynamically expressed in the developing kernel, which will help to exploit new players functioning in maize kernel development.
RESUMEN
Long non-coding RNAs (lncRNAs) have been reported to be involved in the development of maize plant. However, few focused on seed development of maize. Here, we identified 753 lncRNA candidates in maize genome from six seed samples. Similar to the mRNAs, lncRNAs showed tissue developmental stage specific and differential expression, indicating their putative role in seed development. Increasing evidence shows that crosstalk among RNAs mediated by shared microRNAs (miRNAs) represents a novel layer of gene regulation, which plays important roles in plant development. Functional roles and regulatory mechanisms of lncRNAs as competing endogenous RNAs (ceRNA) in plants, particularly in maize seed development, are unclear. We combined analyses of consistently altered 17 lncRNAs, 840 mRNAs and known miRNA to genome-wide investigate potential lncRNA-mediated ceRNA based on "ceRNA hypothesis". The results uncovered seven novel lncRNAs as potential functional ceRNAs. Functional analyses based on their competitive coding-gene partners by Gene Ontology (GO) and KEGG biological pathway demonstrated that combined effects of multiple ceRNAs can have major impacts on general developmental and metabolic processes in maize seed. These findings provided a useful platform for uncovering novel mechanisms of maize seed development and may provide opportunities for the functional characterization of individual lncRNA in future studies.
RESUMEN
The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes l-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αß-ßα sandwich fold homodimer that cleaves its own peptide backbone at the G167-T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167-T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure-activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.
Asunto(s)
Asparaginasa/química , Asparaginasa/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Multimerización de Proteína/fisiología , Dominio Catalítico/fisiología , Cristalografía por Rayos X , Humanos , Estructura Secundaria de ProteínaRESUMEN
A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML-RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Leucémica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Leucemia Promielocítica Aguda/metabolismo , Isomerasa de Peptidilprolil/genética , Tretinoina/metabolismo , Animales , Antineoplásicos/química , Neoplasias de la Mama/genética , Catálisis , Dominio Catalítico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/metabolismo , Células HEK293 , Humanos , Leucemia Promielocítica Aguda/genética , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Peptidilprolil Isomerasa de Interacción con NIMA , Trasplante de Neoplasias , Fosfatos/química , Fosforilación , Prolina/química , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
The unique proline isomerase Pin1 is pivotal for protecting against age-dependent neurodegeneration in Alzheimer's disease (AD), with its inhibition providing a molecular link between tangle and plaque pathologies. Pin1 is oxidatively modified in human AD brains, but little is known about its regulatory mechanisms and pathological significance of such Pin1 modification. In this paper, our determination of crystal structures of oxidized Pin1 reveals a series of Pin1 oxidative modifications on Cys113 in a sequential fashion. Cys113 oxidization is further confirmed by generating antibodies specifically recognizing oxidized Cys113 of Pin1. Furthermore, Pin1 oxidation on Cys113 inactivates its catalytic activity in vitro, and Ala point substitution of Cys113 inactivates the ability of Pin1 to isomerize tau as well as to promote protein turnover of tau and APP. Moreover, redox regulation affects Pin1 subcellular localization and Pin1-mediated neuronal survival in response to hypoxia treatment. Importantly, Cys113-oxidized Pin1 is significantly increased in human AD brain comparing to age-matched controls. These results not only identify a novel Pin1 oxidation site to be the critical catalytic residue Cys113, but also provide a novel oxidative regulation mechanism for inhibiting Pin1 activity in AD. These results suggest that preventing Pin1 oxidization might help to reduce the risk of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Anticuerpos , Dominio Catalítico , Línea Celular Tumoral , Hipocampo/metabolismo , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA , Oxidación-Reducción , Isomerasa de Peptidilprolil/inmunología , Proteínas tau/metabolismoRESUMEN
Ultraviolet photodissociation (UVPD) mass spectrometry (MS) was used to characterize the sequences of proteins in native protein-ligand and protein-protein complexes and to provide auxiliary information about the binding sites of the ligands and protein-protein interfaces. UVPD outperformed collisional induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) in terms of yielding the most comprehensive diagnostic primary sequence information about the proteins in the complexes. UVPD also generated noncovalent fragment ions containing a portion of the protein still bound to the ligand which revealed some insight into the nature of the binding sites of myoglobin/heme, eIF4E/m(7)GTP, and human peptidyl-prolyl cis-trans isomerase 1 (Pin1) in complex with the peptide derived from the C-terminal domain of RNA polymerase II (CTD). Noncovalently bound protein-protein fragment ions from oligomeric ß-lactoglobulin dimers and hexameric insulin complexes were also produced upon UVPD, providing some illumination of tertiary and quaternary protein structural features.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/metabolismo , Hemo/química , Hemo/metabolismo , Caballos , Humanos , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mioglobina/química , Mioglobina/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Procesos Fotoquímicos , Unión Proteica , Multimerización de Proteína , Rayos UltravioletaRESUMEN
The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer's disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissociation methods.
Asunto(s)
Isomerasa de Peptidilprolil/análisis , Proteómica , Rayos Ultravioleta , Humanos , Espectrometría de Masas , Modelos Moleculares , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genética , Procesos FotoquímicosRESUMEN
The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50%), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like hASRGL1-Thr168Ala variant. Crystallographic and biochemical evidence suggest an activation mechanism where a torsional restraint on the Thr168 side chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation.
Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/metabolismo , Asparaginasa/química , Asparaginasa/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Amidohidrolasas/genética , Asparaginasa/genética , Asparagina/metabolismo , Autoantígenos/genética , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Humanos , Hidrólisis , Modelos Moleculares , Mutación Puntual , Conformación ProteicaRESUMEN
The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.
Asunto(s)
Proteínas Bacterianas/química , Vías Biosintéticas/fisiología , Isomerasas/química , Homología Estructural de Proteína , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/fisiología , Benzodiazepinonas/síntesis química , Benzodiazepinonas/química , Benzodiazepinonas/metabolismo , Cristalografía por Rayos X , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Isomerasas/biosíntesis , Isomerasas/fisiología , Cinética , Estructura Terciaria de Proteína/fisiología , Pseudomonas putida/enzimología , Transducción de Señal/fisiología , Staphylococcus aureus/enzimología , Especificidad por Sustrato/fisiologíaRESUMEN
This paper describes the role of alpha-subunit VISIT-DG sequence residues alphaSer-347 and alphaGly-351 in catalytic sites of Escherichia coli F(1)F(o) ATP synthase. X-ray structures show the very highly conserved alpha-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues alphaArg-376, betaArg-182, betaLys-155, and betaArg-246 in the phosphate-binding subdomain. Mutations alphaS347Q and alphaG351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F(1)F(o) in membranes by 100- and 150-fold, respectively, whereas alphaS347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of alphaS347Q mutant was not inhibited, and the alphaS347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and alphaG351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and alphaG351Q mutant ATPase essentially completely, ATPase in alphaS347A or alphaS347Q mutant was inhibited maximally by approximately 80-90%, although reaction still occurred at residue betaTyr-297, proximal to the alpha-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts inbetaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and alphaG351Q mutant but not in alphaS347Q or alphaS347A mutant. The results demonstrate that alphaSer-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, alphaGly-351, although strongly conserved and clearly important for function, appears not to play a direct role.