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1.
BMC Microbiol ; 24(1): 125, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622505

RESUMO

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.


Assuntos
Bacillus subtilis , Ácido Glutâmico , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ácido Glutâmico/metabolismo , Sequência de Aminoácidos , Hidrolases/metabolismo , Ácido Poliglutâmico/genética , Genômica
2.
Biosens Bioelectron ; 255: 116264, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38588629

RESUMO

Chemical-nose strategy has achieved certain success in the discrimination and identification of pathogens. However, this strategy usually relies on non-specific interactions, which are prone to be significantly disturbed by the change of environment thus limiting its practical usefulness. Herein, we present a novel chemical-nose sensing approach leveraging the difference in the dynamic metabolic variation during peptidoglycan metabolism among different species for rapid pathogen discrimination. Pathogens were first tethered with clickable handles through metabolic labeling at two different acidities (pH = 5 and 7) for 20 and 60 min, respectively, followed by click reaction with fluorescence up-conversion nanoparticles to generate a four-dimensional signal output. This discriminative multi-dimensional signal allowed eight types of model bacteria to be successfully classified within the training set into strains, genera, and Gram phenotypes. As the difference in signals of the four sensing channels reflects the difference in the amount/activity of enzymes involved in metabolic labeling, this strategy has good anti-interference capability, which enables precise pathogen identification within 2 h with 100% accuracy in spiked urinary samples and allows classification of unknown species out of the training set into the right phenotype. The robustness of this approach holds significant promise for its widespread application in pathogen identification and surveillance.


Assuntos
Técnicas Biossensoriais , Nanopartículas , Bactérias , Hidrolases , Aprendizado de Máquina
3.
J Agric Food Chem ; 72(14): 8140-8148, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38563232

RESUMO

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.


Assuntos
Diterpenos do Tipo Caurano , Stevia , Trissacarídeos , Saccharomyces cerevisiae/genética , Difosfato de Uridina , Hidrolases , Glucosídeos , Glicosiltransferases/genética , Glicosídeos , Folhas de Planta
4.
PLoS One ; 19(4): e0300767, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38578733

RESUMO

Semantic segmentation of cityscapes via deep learning is an essential and game-changing research topic that offers a more nuanced comprehension of urban landscapes. Deep learning techniques tackle urban complexity and diversity, which unlocks a broad range of applications. These include urban planning, transportation management, autonomous driving, and smart city efforts. Through rich context and insights, semantic segmentation helps decision-makers and stakeholders make educated decisions for sustainable and effective urban development. This study investigates an in-depth exploration of cityscape image segmentation using the U-Net deep learning model. The proposed U-Net architecture comprises an encoder and decoder structure. The encoder uses convolutional layers and down sampling to extract hierarchical information from input images. Each down sample step reduces spatial dimensions, and increases feature depth, aiding context acquisition. Batch normalization and dropout layers stabilize models and prevent overfitting during encoding. The decoder reconstructs higher-resolution feature maps using "UpSampling2D" layers. Through extensive experimentation and evaluation of the Cityscapes dataset, this study demonstrates the effectiveness of the U-Net model in achieving state-of-the-art results in image segmentation. The results clearly shown that, the proposed model has high accuracy, mean IOU and mean DICE compared to existing models.


Assuntos
Aprendizado Profundo , Semântica , Planejamento de Cidades , Pesquisa Empírica , Hidrolases , Processamento de Imagem Assistida por Computador
5.
Molecules ; 29(7)2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38611715

RESUMO

The plant-derived toxin ricin is classified as a type 2 ribosome-inactivating protein (RIP) and currently lacks effective clinical antidotes. The toxicity of ricin is mainly due to its ricin toxin A chain (RTA), which has become an important target for drug development. Previous studies have identified two essential binding pockets in the active site of RTA, but most existing inhibitors only target one of these pockets. In this study, we used computer-aided virtual screening to identify a compound called RSMI-29, which potentially interacts with both active pockets of RTA. We found that RSMI-29 can directly bind to RTA and effectively attenuate protein synthesis inhibition and rRNA depurination induced by RTA or ricin, thereby inhibiting their cytotoxic effects on cells in vitro. Moreover, RSMI-29 significantly reduced ricin-mediated damage to the liver, spleen, intestine, and lungs in mice, demonstrating its detoxification effect against ricin in vivo. RSMI-29 also exhibited excellent drug-like properties, featuring a typical structural moiety of known sulfonamides and barbiturates. These findings suggest that RSMI-29 is a novel small-molecule inhibitor that specifically targets ricin toxin A chain, providing a potential therapeutic option for ricin intoxication.


Assuntos
Ricina , Animais , Camundongos , Proteínas Inativadoras de Ribossomos Tipo 2 , Desenvolvimento de Medicamentos , Hidrolases , Fígado
6.
Molecules ; 29(7)2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38611779

RESUMO

Drug discovery involves a crucial step of optimizing molecules with the desired structural groups. In the domain of computer-aided drug discovery, deep learning has emerged as a prominent technique in molecular modeling. Deep generative models, based on deep learning, play a crucial role in generating novel molecules when optimizing molecules. However, many existing molecular generative models have limitations as they solely process input information in a forward way. To overcome this limitation, we propose an improved generative model called BD-CycleGAN, which incorporates BiLSTM (bidirectional long short-term memory) and Mol-CycleGAN (molecular cycle generative adversarial network) to preserve the information of molecular input. To evaluate the proposed model, we assess its performance by analyzing the structural distribution and evaluation matrices of generated molecules in the process of structural transformation. The results demonstrate that the BD-CycleGAN model achieves a higher success rate and exhibits increased diversity in molecular generation. Furthermore, we demonstrate its application in molecular docking, where it successfully increases the docking score for the generated molecules. The proposed BD-CycleGAN architecture harnesses the power of deep learning to facilitate the generation of molecules with desired structural features, thus offering promising advancements in the field of drug discovery processes.


Assuntos
Fármacos Anti-HIV , Simulação de Acoplamento Molecular , Descoberta de Drogas , Hidrolases , Memória de Longo Prazo
7.
Sci Rep ; 14(1): 8487, 2024 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-38605059

RESUMO

Breast cancer has rapidly increased in prevalence in recent years, making it one of the leading causes of mortality worldwide. Among all cancers, it is by far the most common. Diagnosing this illness manually requires significant time and expertise. Since detecting breast cancer is a time-consuming process, preventing its further spread can be aided by creating machine-based forecasts. Machine learning and Explainable AI are crucial in classification as they not only provide accurate predictions but also offer insights into how the model arrives at its decisions, aiding in the understanding and trustworthiness of the classification results. In this study, we evaluate and compare the classification accuracy, precision, recall, and F1 scores of five different machine learning methods using a primary dataset (500 patients from Dhaka Medical College Hospital). Five different supervised machine learning techniques, including decision tree, random forest, logistic regression, naive bayes, and XGBoost, have been used to achieve optimal results on our dataset. Additionally, this study applied SHAP analysis to the XGBoost model to interpret the model's predictions and understand the impact of each feature on the model's output. We compared the accuracy with which several algorithms classified the data, as well as contrasted with other literature in this field. After final evaluation, this study found that XGBoost achieved the best model accuracy, which is 97%.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Teorema de Bayes , Bangladesh/epidemiologia , Mama , Aprendizado de Máquina , Hidrolases
8.
BMC Biol ; 22(1): 73, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38561772

RESUMO

BACKGROUND: Quorum sensing (QS) is the ability of microorganisms to assess local clonal density by measuring the extracellular concentration of signal molecules that they produce and excrete. QS is also the only known way of bacterial communication that supports the coordination of within-clone cooperative actions requiring a certain threshold density of cooperating cells. Cooperation aided by QS communication is sensitive to cheating in two different ways: laggards may benefit from not investing in cooperation but enjoying the benefit provided by their cooperating neighbors, whereas Liars explicitly promise cooperation but fail to do so, thereby convincing potential cooperating neighbors to help them, for almost free. Given this double vulnerability to cheats, it is not trivial why QS-supported cooperation is so widespread among prokaryotes. RESULTS: We investigated the evolutionary dynamics of QS in populations of cooperators for whom the QS signal is an inevitable side effect of producing the public good itself (cue-based QS). Using spatially explicit agent-based lattice simulations of QS-aided threshold cooperation (whereby cooperation is effective only above a critical cumulative level of contributions) and three different (analytical and numerical) approximations of the lattice model, we explored the dynamics of QS-aided threshold cooperation under a feasible range of parameter values. We demonstrate three major advantages of cue-driven cooperation. First, laggards cannot wipe out cooperation under a wide range of reasonable environmental conditions, in spite of an unconstrained possibility to mutate to cheating; in fact, cooperators may even exclude laggards at high cooperation thresholds. Second, lying almost never pays off, if the signal is an inevitable byproduct (i.e., the cue) of cooperation; even very cheap fake signals are selected against. And thirdly, QS is most useful if local cooperator densities are the least predictable, i.e., if their lattice-wise mean is close to the cooperation threshold with a substantial variance. CONCLUSIONS: Comparing the results of the four different modeling approaches indicates that cue-driven threshold cooperation may be a viable evolutionary strategy for microbes that cannot keep track of past behavior of their potential cooperating partners, in spatially viscous and in well-mixed environments alike. Our model can be seen as a version of the famous greenbeard effect, where greenbeards coexist with defectors in a evolutionarily stable polymorphism. Such polymorphism is maintained by the condition-dependent trade-offs of signal production which are characteristic of cue-based QS.


Assuntos
Sinais (Psicologia) , Percepção de Quorum , Evolução Biológica , Bactérias , Hidrolases , Comunicação
9.
Biotechnol J ; 19(4): e2400053, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38593303

RESUMO

The rapid escalation of plastic waste accumulation presents a significant threat of the modern world, demanding an immediate solution. Over the last years, utilization of the enzymatic machinery of various microorganisms has emerged as an environmentally friendly asset in tackling this pressing global challenge. Thus, various hydrolases have been demonstrated to effectively degrade polyesters. Plastic waste streams often consist of a variety of different polyesters, as impurities, mainly due to wrong disposal practices, rendering recycling process challenging. The elucidation of the selective degradation of polyesters by hydrolases could offer a proper solution to this problem, enhancing the recyclability performance. Towards this, our study focused on the investigation of four bacterial polyesterases, including DaPUase, IsPETase, PfPHOase, and Se1JFR, a novel PETase-like lipase. The enzymes, which were biochemically characterized and structurally analyzed, demonstrated degradation ability of synthetic plastics. While a consistent pattern of polyesters' degradation was observed across all enzymes, Se1JFR stood out in the degradation of PBS, PLA, and polyether PU. Additionally, it exhibited comparable results to IsPETase, a benchmark mesophilic PETase, in the degradation of PCL and semi-crystalline PET. Our results point out the wide substrate spectrum of bacterial hydrolases and underscore the significant potential of PETase-like enzymes in polyesters degradation.


Assuntos
Hidrolases , Poliésteres , Hidrolases/metabolismo , Poliésteres/química , Bactérias/metabolismo , Lipase , Polietilenotereftalatos/química
10.
Eur Rev Med Pharmacol Sci ; 28(5): 1741-1750, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38497856

RESUMO

OBJECTIVE: To evaluate the prevalence of bifid mandibular canals (BMC) using cone-beam computed tomography (CBCT) in the Saudi population subset. MATERIALS AND METHODS: In the study, three hundred and forty-three CBCT scans (661 sides) were evaluated for the presence of BMC, involving 162 males and 181 females. Tomographic acquisitions were performed on the device Planmeca®. The image analysis was performed on the Planmeca Romexis® software, aided by image filters associated with transverse, oblique, and panoramic reconstruction cuts for analysis of the BMC. Naitoh's classification (2009) was employed to classify mandibular canals. The prevalence of BMC was determined according to location, gender, and age of participants. The data were analyzed with Chi-square and one-way ANOVA tests at a significance level of 95%. RESULTS: The BMC was observed in 37 (12.34%) out of 343 participants, of whom 20 (54.05%) were males and 17 (45.94%) were females. There was no significant difference in the proportion of bifid canals in both genders and various age groups. The most common BMC was the retromolar canal type, with 56.75% occurrence. The dental canal type was observed in 18.91% of BMC participants. The presence of a forward canal without confluence was observed in 16.21% of participants in the BMC, whereas a forward canal with confluence was noted in 8.10% of participants. CONCLUSIONS: The prevalence of bifid mandibular canals (BMC) within a subset of the Saudi population was 12.3%. The retromolar canal was identified as the most frequently occurring type, accounting for 56.7% of cases. No significant variations in BMC prevalence were observed concerning age and gender. Consequently, it is strongly advised to conduct a thorough assessment of the mandibular canal and its potential variations using CBCT imaging before undertaking mandibular surgical procedures, in order to minimize the risk of complications.


Assuntos
Tomografia Computadorizada de Feixe Cônico , Canal Mandibular , Humanos , Feminino , Masculino , Estudos Retrospectivos , Hidrolases , Processamento de Imagem Assistida por Computador
11.
Elife ; 122024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38488661

RESUMO

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Replicação do DNA , Proteínas de Ligação a DNA , Humanos , ATPases Associadas a Diversas Atividades Celulares/metabolismo , DNA , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Hidrolases/genética , Dedos de Zinco
12.
PLoS One ; 19(3): e0297561, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38498552

RESUMO

This study aims to advance the Multilevel Analysis of Individual Heterogeneity and Discriminatory Accuracy (MAIHDA) approach by addressing two key questions. First, it investigates the impact of using increasingly complex combinations of variables to create intersectional strata on between-stratum variance, measured by the variance partitioning coefficients (VPCs). Second, it examines the stability of coefficients for fixed effects across models with an increasing number of hierarchical levels. The analysis is performed using data from a survey of over 42,000 respondents on the prevalence of gender-based violence in European research organisations conducted in 2022. Results indicate that the number of intersectional strata is not significantly related to the proportion of the total variance attributable to the variance between intersectional strata in the MAIHDA approach. Moreover, the coefficients remain relatively stable and consistent across models with increasing complexity, where levels about organisations and countries are added. The analysis concludes that the MAIHDA approach can be flexibly applied for different research purposes, either to better account for structures of power and inequality; or to provide intersectionality-sensitive estimates. The findings underscore the need for researchers to clarify the specific aims of using MAIHDA, whether descriptive or inferential, and highlight the approach's versatility in addressing intersectionality within quantitative research. The study contributes to the literature by offering empirical evidence on the methodological considerations in applying the MAIHDA approach, thereby aiding in its more effective use for intersectional research.


Assuntos
Violência de Gênero , Análise Multinível , Hidrolases , Enquadramento Interseccional
13.
Nat Commun ; 15(1): 2452, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38503748

RESUMO

ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.


Assuntos
Legionella , Ubiquitina , Animais , Ubiquitina/metabolismo , Legionella/metabolismo , Drosophila melanogaster/metabolismo , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Hidrolases/metabolismo
14.
Sci Rep ; 14(1): 6778, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514802

RESUMO

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance (19F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed ß-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using 19F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis.


Assuntos
Glucose , Hidrolases , Melhoramento Vegetal , Ácidos Indolacéticos/química , Indóis , Grão Comestível
15.
Cell Rep ; 43(3): 113942, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38489266

RESUMO

Tumor-associated macrophages (TAMs) shape tumor immunity and therapeutic efficacy. However, it is poorly understood whether and how post-translational modifications (PTMs) intrinsically affect the phenotype and function of TAMs. Here, we reveal that peptidylarginine deiminase 4 (PAD4) exhibits the highest expression among common PTM enzymes in TAMs and negatively correlates with the clinical response to immune checkpoint blockade. Genetic and pharmacological inhibition of PAD4 in macrophages prevents tumor progression in tumor-bearing mouse models, accompanied by an increase in macrophage major histocompatibility complex (MHC) class II expression and T cell effector function. Mechanistically, PAD4 citrullinates STAT1 at arginine 121, thereby promoting the interaction between STAT1 and protein inhibitor of activated STAT1 (PIAS1), and the loss of PAD4 abolishes this interaction, ablating the inhibitory role of PIAS1 in the expression of MHC class II machinery in macrophages and enhancing T cell activation. Thus, the PAD4-STAT1-PIAS1 axis is an immune restriction mechanism in macrophages and may serve as a cancer immunotherapy target.


Assuntos
Hidrolases , Processamento de Proteína Pós-Traducional , Camundongos , Animais , Desiminases de Arginina em Proteínas/metabolismo , Proteína-Arginina Desiminase do Tipo 4/genética , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Hidrolases/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Macrófagos/metabolismo
16.
Sci Adv ; 10(13): eadk7201, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38536910

RESUMO

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Cristalografia por Raios X , Proteínas/química , Catálise , Conformação Proteica , Hidrolases
17.
J Hazard Mater ; 469: 133967, 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38457978

RESUMO

Diclofop-methyl, an aryloxyphenoxypropionate (AOPP) herbicide, is a chiral compound with two enantiomers. Microbial detoxification and degradation of various enantiomers is garnering immense research attention. However, enantioselective catabolism of diclofop-methyl has been rarely explored, especially at the molecular level. This study cloned two novel hydrolase genes (dcmA and dcmH) in Sphingopyxis sp. DBS4, and characterized them for diclofop-methyl degradation. DcmA, a member of the amidase superfamily, exhibits 26.1-45.9% identity with functional amidases. Conversely, DcmH corresponded to the DUF3089 domain-containing protein family (a family with unknown function), sharing no significant similarity with other biochemically characterized proteins. DcmA exhibited a broad spectrum of substrates, with preferential hydrolyzation of (R)-(+)-diclofop-methyl, (R)-(+)-quizalofop-ethyl, and (R)-(+)-haloxyfop-methyl. DcmH also preferred (R)-(+)-quizalofop-ethyl and (R)-(+)-haloxyfop-methyl degradation while displaying no apparent enantioselective activity towards diclofop-methyl. Using site-directed mutagenesis and molecular docking, it was determined that Ser175 was the fundamental residue influencing DcmA's activity against the two enantiomers of diclofop-methyl. For the degradation of AOPP herbicides, DcmA is an enantioselective amidase that has never been reported in research. This study provided novel hydrolyzing enzyme resources for the remediation of diclofop-methyl in the environment and deepened the understanding of enantioselective degradation of chiral AOPP herbicides mediated by microbes.


Assuntos
Éteres Difenil Halogenados , Herbicidas , Maleatos , Propionatos , Quinoxalinas , Herbicidas/metabolismo , Hidrolases , Simulação de Acoplamento Molecular , Estereoisomerismo , Produtos da Oxidação Avançada de Proteínas
18.
Bioresour Technol ; 399: 130549, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38461869

RESUMO

The biogas upgrading process through bioconversion of CO2 to CH4 by hydrogenotrophic methanogens is an attractive strategy for energy decarbonation. Many studies have optimized operational parameters to improve key performance indicators such as CH4% and H2 utilization efficiency. However, inconsistent laboratory conditions make it challenging to compare results. Existing models for analyzing operating conditions can only assess the impact of individual conditions and lack the ability to simultaneously optimize multiple conditions. To address this, two XGBoost models were built with R2 of 0.779 and 0.903 with data collected from literatures and were embedded into multi-objective partitive swarm optimization algorithm to optimal operating conditions. Predictions were compared with experimental validations under optimized conditions, revealing an 8.50% and 2.95% relative error in CH4% and H2 conversion rate, respectively. This approach streamlines biogas upgrading processes, offering a data-driven solution to enhance efficiency and consistency in the pursuit of sustainable methane production.


Assuntos
Biocombustíveis , Reatores Biológicos , Dióxido de Carbono , Metano , Hidrogênio , Hidrolases
19.
Int J Mol Sci ; 25(6)2024 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-38542155

RESUMO

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.


Assuntos
Actinas , Citrulinação , Camundongos , Animais , Actinas/metabolismo , Tubulina (Proteína)/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Citrulina/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hidrolases/metabolismo
20.
J Agric Food Chem ; 72(13): 7279-7290, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38519413

RESUMO

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze ß-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.


Assuntos
Lactococcus lactis , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Sequência de Aminoácidos , Dipeptidil Peptidases e Tripeptidil Peptidases , Peptídeos/genética , Hidrolases , Aminopeptidases/genética , Aminopeptidases/química , Aminopeptidases/metabolismo , Concentração de Íons de Hidrogênio
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