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1.
Appl Microbiol Biotechnol ; 108(1): 404, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38953996

RESUMO

Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals. KEY POINTS: • PET hydrolases must be thermophilic, but their operation must be below 70 °C • Classical and state-of-the-art engineering approaches are useful for PET hydrolases • Enzyme activity on crystalline PET is most expected for future PET biorecycling.


Assuntos
Hidrolases , Polietilenotereftalatos , Polietilenotereftalatos/metabolismo , Polietilenotereftalatos/química , Hidrolases/metabolismo , Hidrolases/química , Hidrolases/genética , Hidrólise , Engenharia de Proteínas/métodos , Biodegradação Ambiental , Reciclagem
2.
MAbs ; 16(1): 2375798, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38984665

RESUMO

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.


Assuntos
Anticorpos Monoclonais , Cricetulus , Hidrolases , Células CHO , Animais , Anticorpos Monoclonais/química , Hidrolases/metabolismo , Polissorbatos/química , Produtos Biológicos/metabolismo , Humanos
3.
Protein Sci ; 33(8): e5122, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39031458

RESUMO

Enterobactin is a high-affinity iron chelator produced and secreted by Escherichia coli and Salmonella typhimurium to scavenge scarce extracellular Fe3+ as a micronutrient. EntC and EntB are the first two enzymes in the enterobactin biosynthetic pathway. Isochorismate, produced by EntC, is a substrate for EntB isochorismatase. By using a competing isochorismate-consuming enzyme (the E. coli SEPHCHC synthase MenD), we found in a coupled assay that residual EntB isochorismatase activity decreased as a function of increasing MenD concentration. In the presence of excess MenD, EntB isochorismatase activity was observed to decrease by 84%, indicative of partial EntC-EntB channeling (16%) of isochorismate. Furthermore, addition of glycerol to the assay resulted in an increase of residual EntB isochorismatase activity to approximately 25% while in the presence of excess MenD. These experimental outcomes supported the existence of a substrate channeling surface identified in a previously reported protein-docking model of the EntC-EntB complex. Two positively charged EntB residues (K21 and R196) that were predicted to electrostatically guide negatively charged isochorismate between the EntC and EntB active sites were mutagenized to determine their effects on substrate channeling. The EntB variants K21D and R196D exhibited a near complete loss of isochorismatase activity, likely due to electrostatic repulsion of the negatively charged isochorismate substrate. Variants K21A, R196A, and K21A/R196A retained partial EntB isochorismatase activity in the absence of EntC; in the presence of EntC, isochorismatase activity in all variants increased to near wild-type levels. The MenD competition assay of the variants revealed that while K21A channeled isochorismate as efficiently as wild-type EntB (~ 15%), the variants K21A/R196A and R196A exhibited an approximately 5-fold loss in observed channeling efficiency (~3%). Taken together, these results demonstrate that partial substrate channeling occurs between EntC and EntB via a leaky electrostatic tunnel formed upon dynamic EntC-EntB complex formation and that EntB R196 plays an essential role in isochorismate channeling.


Assuntos
Enterobactina , Proteínas de Escherichia coli , Escherichia coli , Enterobactina/biossíntese , Enterobactina/metabolismo , Enterobactina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ácido Corísmico/metabolismo , Ácido Corísmico/química , Hidrolases
4.
Nat Commun ; 15(1): 6121, 2024 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-39033196

RESUMO

The biguanide drug metformin is a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the global environment. However, little is known about the fate of metformin by microbial catabolism. Here, we characterize a Ni2+-dependent heterohexameric enzyme (MetCaCb) from the ureohydrolase superfamily, catalyzing the hydrolysis of metformin into guanylurea and dimethylamine. Either subunit alone is catalytically inactive, but together they work as an active enzyme highly specific for metformin. The crystal structure of the MetCaCb complex shows the coordination of the binuclear metal cluster only in MetCa, with MetCb as a protein binder of its active cognate. An in-silico search and functional assay discover a group of MetCaCb-like protein pairs exhibiting metformin hydrolase activity in the environment. Our findings not only establish the genetic and biochemical foundation for metformin catabolism but also provide additional insights into the adaption of the ancient enzymes toward newly occurred substrate.


Assuntos
Hidrolases , Metformina , Níquel , Metformina/metabolismo , Metformina/química , Níquel/metabolismo , Níquel/química , Hidrolases/metabolismo , Hidrolases/química , Hidrolases/genética , Cristalografia por Raios X , Hidrólise , Especificidade por Substrato , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares
5.
Anal Biochem ; 693: 115598, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38964700

RESUMO

The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolyzing polyamides have been proposed as biocatalysts that may be suitable for this application. NylC is an enzyme that can mediate the hydrolysis of aminohexanoic acid oligomers, and to some extent, bulk polymers. However, current assays to characterize the activity of this enzyme require long reaction times and/or rely on secondary reactions to quantify hydrolysis. Herein, we have designed structurally-optimized small molecule chromogenic esters that serve as substrate analogues for monitoring NylC acyltransferase activity in a continuous manner. This assay can be performed in minutes at room temperature, and the substrate N-acetyl-GABA-pNP ester (kcat = 0.37 s-1, KM = 256 µM) shows selectivity for NylC in complex biological media. We also demonstrate that activity towards this substrate analogue correlates with amide hydrolysis, which is the primary activity of this enzyme. Furthermore, our screening of substrate analogues provides insight into the substrate specificity of NylC, which is relevant to biocatalytic applications.


Assuntos
Nylons , Nylons/química , Nylons/metabolismo , Hidrólise , Especificidade por Substrato , Hidrolases/metabolismo , Hidrolases/química , Aciltransferases/metabolismo , Aciltransferases/química , Aciltransferases/análise , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química
6.
J Assoc Physicians India ; 72(5): 97-100, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38881119

RESUMO

Chorea is a very commonly encountered movement disorder; it has various etiologies, and it can have autoimmune, vascular, degenerative, or paraneoplastic etiology. Our patient had acute onset chorea and a strong history of smoking, which made us suspect first vascular followed by paraneoplastic cause. After ruling out common vascular and metabolic causes, his whole body positron emission tomography (PET) scan revealed a mass in the right upper lobe, a biopsy revealed a small cell carcinoma lung and a paraneoplastic panel showed antibodies positive for collapsin response mediator protein 5 antigen (CRMP-5/CV2); the patient was started on immunomodulation, chemotherapy with the variable response, he succumbed to a cardiac event after treatment.


Assuntos
Coreia , Neoplasias Pulmonares , Humanos , Coreia/etiologia , Coreia/diagnóstico , Masculino , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/diagnóstico , Proteínas do Tecido Nervoso/imunologia , Carcinoma de Pequenas Células do Pulmão/complicações , Evolução Fatal , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Hidrolases , Proteínas Associadas aos Microtúbulos
7.
Front Cell Infect Microbiol ; 14: 1392249, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38915922

RESUMO

In recent years, there has been increasing interest in studying gut microbiome-derived hydrolases in relation to oral drug metabolism, particularly focusing on natural product drugs. Despite the significance of natural product drugs in the field of oral medications, there is a lack of research on the regulatory interplay between gut microbiome-derived hydrolases and these drugs. This review delves into the interaction between intestinal microbiome-derived hydrolases and natural product drugs metabolism from three key perspectives. Firstly, it examines the impact of glycoside hydrolases, amide hydrolases, carboxylesterase, bile salt hydrolases, and epoxide hydrolase on the structure of natural products. Secondly, it explores how natural product drugs influence microbiome-derived hydrolases. Lastly, it analyzes the impact of interactions between hydrolases and natural products on disease development and the challenges in developing microbial-derived enzymes. The overarching goal of this review is to lay a solid theoretical foundation for the advancement of research and development in new natural product drugs and personalized treatment.


Assuntos
Produtos Biológicos , Microbioma Gastrointestinal , Hidrolases , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Humanos , Hidrolases/metabolismo , Animais , Glicosídeo Hidrolases/metabolismo , Bactérias/metabolismo , Bactérias/enzimologia
8.
Int J Mol Sci ; 25(11)2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38891866

RESUMO

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.


Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Hidrolases , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Camundongos , Hidrolases/metabolismo , Hidrolases/genética , Regiões Promotoras Genéticas , Óperon/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Vibrio/genética , Vibrio/metabolismo , Vibrio/patogenicidade , Arginina/metabolismo , Família Multigênica , Virulência/genética , Viabilidade Microbiana
9.
Mol Biol Cell ; 35(8): mr6, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38888935

RESUMO

Maintenance of a pool of active lysosomes with acidic pH and degradative hydrolases is crucial for cell health. Abnormalities in lysosomal function are closely linked to diseases, such as lysosomal storage disorders, neurodegeneration, intracellular infections, and cancer among others. Emerging body of research suggests the malfunction of lysosomal hydrolase trafficking pathway to be a common denominator of several disease pathologies. However, available conventional tools to assess lysosomal hydrolase trafficking are insufficient and fail to provide a comprehensive picture about the trafficking flux and location of lysosomal hydrolases. To address some of the shortcomings, we designed a genetically-encoded fluorescent reporter containing a lysosomal hydrolase tandemly tagged with pH sensitive and insensitive fluorescent proteins, which can spatiotemporally trace the trafficking of lysosomal hydrolases. As a proof of principle, we demonstrate that the reporter can detect perturbations in hydrolase trafficking, that are induced by pharmacological manipulations and pathophysiological conditions like intracellular protein aggregates. This reporter can effectively serve as a probe for mapping the mechanistic intricacies of hydrolase trafficking pathway in health and disease and is a utilitarian tool to identify genetic and pharmacological modulators of this pathway, with potential therapeutic implications.


Assuntos
Hidrolases , Lisossomos , Manosefosfatos , Transporte Proteico , Humanos , Lisossomos/metabolismo , Manosefosfatos/metabolismo , Hidrolases/metabolismo , Hidrolases/genética , Fluorescência , Genes Reporter , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Células HeLa
10.
Sci Rep ; 14(1): 14449, 2024 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-38914665

RESUMO

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.


Assuntos
Escherichia coli , Robótica , Robótica/métodos , Escherichia coli/genética , Engenharia de Proteínas/métodos , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/economia , Hidrolases/metabolismo , Hidrolases/química , Hidrolases/genética , Polietilenotereftalatos/química , Reprodutibilidade dos Testes
11.
Arch Biochem Biophys ; 758: 110048, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38848996

RESUMO

The inherent structural properties of enzymes are critical in defining catalytic function. Often, studies to evaluate the relationship between structure and function are limited to only one defined structural element. The two-component flavin-dependent desulfonase family of enzymes involved in bacterial sulfur acquisition utilize a comprehensive range of structural features to carry out the desulfonation of organosulfur compounds. These metabolically essential two-component FMN-dependent desulfonase systems have been proposed to utilize oligomeric changes, protein-protein interactions for flavin transfer, and common mechanistic steps for carbon-sulfur bond cleavage. This review is focused on our current functional and structural understanding of two-component FMN-dependent desulfonase systems from multiple bacterial sources. Mechanistic and structural comparisons from recent independent studies provide fresh insights into the overall functional properties of these systems and note areas in need of further investigation. The review acknowledges current studies focused on evaluating the structural properties of these enzymes in relationship to their distinct catalytic function. The role of these enzymes in maintaining adequate sulfur levels, coupled with the conserved nature of these enzymes in diverse bacteria, underscore the importance in understanding the functional and structural nuances of these systems.


Assuntos
Proteínas de Bactérias , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bactérias/enzimologia , Compostos de Enxofre/metabolismo , Compostos de Enxofre/química , Hidrolases/química , Hidrolases/metabolismo , Mononucleotídeo de Flavina/metabolismo , Mononucleotídeo de Flavina/química , Enxofre/metabolismo , Enxofre/química , Flavinas/metabolismo , Flavinas/química , Relação Estrutura-Atividade , Carbono/metabolismo , Carbono/química
12.
ACS Appl Mater Interfaces ; 16(27): 35566-35575, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38922631

RESUMO

Encapsulating enzymes within metal-organic frameworks such as zeolitic imidazolate framework-8 (ZIF-8) has been demonstrated to enhance enzymatic performance under harsh conditions. However, by computer-aided analysis, we revealed that highly hydrophobic organic ligands and unfavorable metal ions could greatly impair the activity of haloalkane dehalogenase DhaA by directly interacting with the catalytic sites, causing an extremely low activity of DhaA after encapsulating within ZIF-8. We also found that the presence of a protecting polymer could protect DhaA from the damage of organic ligands and metal ions and that a positively charged amino acid could increase the DhaA activity. Based on the simulations and experimental observations, we have designed to coencapsulate DhaA with poly(vinylpyrrolidone) (PVP) and lysine (Lys) within the amorphous Co-based metal azolate coordination polymer (CoCP). The as-prepared immobilized enzyme (DhaA/PVP/Lys@CoCP) exhibited significantly increased activity (91.5 times higher than that of DhaA@ZIF-8), dramatically enhanced thermostability at 50-70 °C, greatly improved catalytic performance in several organic solvent solutions, and good recyclability (over 75% of the initial activity after 10 cycles). The superiority of the immobilized enzyme was also demonstrated with a substrate frequently detected in the real world. In addition to the protective effect of PVP and positive effect of Lys, experimental and computational investigations unveiled other two favorable aspects that contributed to the enhanced enzymatic performance: (1) high hydrophilicity of the immobilization material and (2) the use of Co2+ with a minimal negative effect on DhaA. The research has thus provided a promising immobilized DhaA with favorable catalytic performance and great potential in industrial applications.


Assuntos
Enzimas Imobilizadas , Hidrolases , Interações Hidrofóbicas e Hidrofílicas , Estruturas Metalorgânicas , Hidrolases/química , Hidrolases/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Estruturas Metalorgânicas/química , Polímeros/química
13.
J Hazard Mater ; 474: 134838, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38850944

RESUMO

Microplastics (MPs) pose an emerging threat to soil ecological function, yet effective solutions remain limited. This study introduces a novel approach using magnetic biochar immobilized PET hydrolase (MB-LCC-FDS) to degrade soil polyethylene terephthalate microplastics (PET-MPs). MB-LCC-FDS exhibited a 1.68-fold increase in relative activity in aquatic solutions and maintained 58.5 % residual activity after five consecutive cycles. Soil microcosm experiment amended with MB-LCC-FDS observed a 29.6 % weight loss of PET-MPs, converting PET into mono(2-hydroxyethyl) terephthalate (MHET). The generated MHET can subsequently be metabolized by soil microbiota to release terephthalic acid. The introduction of MB-LCC-FDS shifted the functional composition of soil microbiota, increasing the relative abundances of Microbacteriaceae and Skermanella while reducing Arthobacter and Vicinamibacteraceae. Metagenomic analysis revealed that MB-LCC-FDS enhanced nitrogen fixation, P-uptake and transport, and organic-P mineralization in PET-MPs contaminated soil, while weakening the denitrification and nitrification. Structural equation model indicated that changes in soil total carbon and Simpson index, induced by MB-LCC-FDS, were the driving factors for soil carbon and nitrogen transformation. Overall, this study highlights the synergistic role of magnetic biochar-immobilized PET hydrolase and soil microbiota in degrading soil PET-MPs, and enhances our understanding of the microbiome and functional gene responses to PET-MPs and MB-LCC-FDS in soil systems.


Assuntos
Carvão Vegetal , Hidrolases , Fósforo , Polietilenotereftalatos , Microbiologia do Solo , Poluentes do Solo , Hidrolases/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Poluentes do Solo/metabolismo , Carvão Vegetal/química , Fósforo/metabolismo , Fósforo/química , Microplásticos/toxicidade , Biodegradação Ambiental , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Nitrogênio/metabolismo , Ciclo do Nitrogênio , Microbiota/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Bactérias/efeitos dos fármacos
14.
Biochemistry ; 63(13): 1663-1673, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38885634

RESUMO

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.


Assuntos
Burkholderiales , Burkholderiales/enzimologia , Burkholderiales/genética , Burkholderiales/metabolismo , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/química , Hidrolases/metabolismo , Hidrolases/genética , Hidrolases/química , Solubilidade , Polietilenotereftalatos/metabolismo , Polietilenotereftalatos/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Engenharia de Proteínas/métodos , Dobramento de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Modelos Moleculares
15.
Commun Biol ; 7(1): 781, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38944651

RESUMO

Macrolide antibiotics, pivotal in clinical therapeutics, are confronting resistance challenges mediated by enzymes like macrolide esterases, which are classified into Ere-type and the less studied Est-type. In this study, we provide the biochemical confirmation of EstX, an Est-type macrolide esterase that initially identified as unknown protein in the 1980s. EstX is capable of hydrolyzing four 16-membered ring macrolides, encompassing both veterinary (tylosin, tidipirosin, and tilmicosin) and human-use (leucomycin A5) antibiotics. It uses typical catalytic triad (Asp233-His261-Ser102) from alpha/beta hydrolase superfamily for ester bond hydrolysis. Further genomic context analysis suggests that the dissemination of estX is likely facilitated by mobile genetic elements such as integrons and transposons. The global distribution study indicates that bacteria harboring the estX gene, predominantly pathogenic species like Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae, are prevalent in 74 countries across 6 continents. Additionally, the emergence timeline of the estX gene suggests its proliferation may be linked to the overuse of macrolide antibiotics. The widespread prevalence and dissemination of Est-type macrolide esterase highlight an urgent need for enhanced monitoring and in-depth research, underlining its significance as an escalating public health issue.


Assuntos
Esterases , Esterases/genética , Esterases/metabolismo , Esterases/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Macrolídeos/metabolismo , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Filogenia , Hidrolases/genética , Hidrolases/metabolismo , Hidrolases/química
16.
J Dent ; 147: 105107, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38830531

RESUMO

OBJECTIVES: To evaluate the effect of daily use of a multiple-enzyme lozenge on de novo plaque formation, on gingivitis development, and on the oral microbiome composition. METHODS: This trial with two parallel arms included 24 healthy adults allocated to the Active (n = 12) or Placebo (n = 12) group. Subjects consumed one lozenge three times daily for seven days, and no oral hygiene procedures were allowed. Differences in de novo plaque accumulation between a baseline period, and one and seven days of intervention were assessed by the Turesky-modification of the Quigley-and-Hein-Plaque-Index (TM-QHPI). The development of gingivitis after seven days of intervention was assessed by the Gingival Index (GI). Plaque and saliva samples were collected at baseline and after seven days of intervention, and evaluated by 16S rRNA gene sequencing. RESULTS: All subjects completed the study, and no adverse events were reported. After one day, the average TM-QHPI was significantly lower in the Active than in the Placebo group, as compared to baseline (p = 0.012). After 7 days, average TM-QHPI values did not differ significantly between groups (p = 0.37). GI values did not increase during the intervention period, with no difference between groups (p = 0.62). Bacterial richness increased in both plaque and saliva samples over a seven-day oral hygiene-free period, with a statistically significant difference for the saliva samples (p = 0.0495) between groups. CONCLUSIONS: A multiple-enzymes lozenge decreased the build-up of de novo plaque after one day and slowed down the process of species increment in saliva. The lozenge may be an adjunct to regular mechanical plaque removal. CLINICAL SIGNIFICANCE: Dental plaque is the main cause of caries, gingivitis, and periodontitis. The search for therapeutic adjuncts to mechanical plaque removal that have no harmful effects on the oral microbiome is important. Treatment with multiple plaque-matrix degrading enzymes is a promising non-biocidal approach to plaque control.


Assuntos
Biofilmes , Índice de Placa Dentária , Placa Dentária , Gengivite , Índice Periodontal , Saliva , Humanos , Placa Dentária/microbiologia , Feminino , Gengivite/microbiologia , Masculino , Biofilmes/efeitos dos fármacos , Adulto , Saliva/microbiologia , Projetos Piloto , Adulto Jovem , RNA Ribossômico 16S , Microbiota/efeitos dos fármacos , Método Duplo-Cego , Higiene Bucal , Resultado do Tratamento , Hidrolases/uso terapêutico , Pessoa de Meia-Idade
17.
J Hazard Mater ; 473: 134716, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38797074

RESUMO

Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 µg/L OTA was completely degraded by 1.0 µg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-ß-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.


Assuntos
Lysobacter , Ocratoxinas , Ocratoxinas/metabolismo , Ocratoxinas/toxicidade , Lysobacter/metabolismo , Lysobacter/genética , Amidoidrolases/metabolismo , Amidoidrolases/genética , Carboxipeptidases/metabolismo , Carboxipeptidases/genética , Hidrolases/metabolismo , Hidrolases/genética
18.
J Chem Inf Model ; 64(11): 4530-4541, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38808649

RESUMO

By performing molecular dynamics (MD), quantum mechanical/molecular mechanical (QM/MM) calculations, and QM cluster calculations, the origin of chemoselectivity of halohydrin dehalogenase (HHDH)-catalyzed ring-opening reactions of epoxide with the nucleophilic reagent NO2- has been explored. Four possible chemoselective pathways were considered, and the computed results indicate that the pathway associated with the nucleophilic attack on the Cα position of epoxide by NO2- is most energetically favorable and has an energy barrier of 12.9 kcal/mol, which is close to 14.1 kcal/mol derived from experimental kinetic data. A hydrogen bonding network formed by residues Ser140, Tyr153, and Arg157 can strengthen the electrophilicity of the active site of the epoxide substrate to affect chemoselectivity. To predict the energy barrier trends of the chemoselective transition states, multiple analyses including distortion analysis and electrophilic Parr function (Pk+) analysis were carried out with or without an enzyme environment. The obtained insights should be valuable for the rational design of enzyme-catalyzed and biomimetic organocatalytic epoxide ring-opening reactions with special chemoselectivity.


Assuntos
Biocatálise , Compostos de Epóxi , Hidrolases , Hidrolases/metabolismo , Hidrolases/química , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Simulação de Dinâmica Molecular , Teoria Quântica , Domínio Catalítico , Especificidade por Substrato
19.
J Hazard Mater ; 472: 134532, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38749251

RESUMO

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.


Assuntos
Fezes , Hidrolases , Polietilenotereftalatos , Streptomyces , Polietilenotereftalatos/metabolismo , Polietilenotereftalatos/química , Streptomyces/enzimologia , Streptomyces/genética , Hidrolases/metabolismo , Hidrolases/genética , Hidrolases/química , Humanos , Fezes/microbiologia , Pseudomonas/enzimologia , Pseudomonas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderiales
20.
Int J Biol Macromol ; 269(Pt 1): 132083, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38705327

RESUMO

Arginine deiminase (ADI) has garnered significant interest because of its ability to objectively eradicate cancer cells and produce L-citrulline. To meet the production demands, this study focused on enhancing the enzyme activity and thermal stability of ADI. In this study, 24 ADI mutants were obtained through computer aid site-specific mutation in the ADI of Enterobacter faecalis. Notably, the specific enzyme activities of F44W, N163P, E220I, E220L, N318E, A336G, T340I, and N382F increased, reaching 1.33-2.53 times that of the original enzyme. This study confirmed that site-specific mutations are critical for optimizing enzyme function. Additionally, the F44W, N163P, E220I, T340I, and A336G mutants demonstrated good thermal stability. The optimal pH for mutant F44W increased to 8, whereas mutants E220I, I244V, A336G, T340I, and N328F maintained an optimal pH of 7.5. Conversely, the M109L, N163P, E220L, I244L, and N318E mutants shad an optimal pH of 7. This study revealed that mutant enzymes with increased activity were more likely to contain mutation sites situated near the four loops associated with catalytic residues, whereas mutations at the dimer junction sites had a higher tendency to enhance enzyme stability. These findings contribute to the development of ADI industrial applications and its modifications.


Assuntos
Estabilidade Enzimática , Hidrolases , Hidrolases/química , Hidrolases/genética , Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Mutação , Cinética , Engenharia de Proteínas/métodos , Biocatálise , Mutagênese Sítio-Dirigida , Modelos Moleculares , Temperatura
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