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1.
Cell Commun Signal ; 22(1): 259, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38715050

RESUMO

Ubiquitination and deubiquitination are important forms of posttranslational modification that govern protein homeostasis. Deubiquitinating enzymes (DUBs), a protein superfamily consisting of more than 100 members, deconjugate ubiquitin chains from client proteins to regulate cellular homeostasis. However, the dysregulation of DUBs is reportedly associated with several diseases, including cancer. The tumor microenvironment (TME) is a highly complex entity comprising diverse noncancerous cells (e.g., immune cells and stromal cells) and the extracellular matrix (ECM). Since TME heterogeneity is closely related to tumorigenesis and immune evasion, targeting TME components has recently been considered an attractive therapeutic strategy for restoring antitumor immunity. Emerging studies have revealed the involvement of DUBs in immune modulation within the TME, including the regulation of immune checkpoints and immunocyte infiltration and function, which renders DUBs promising for potent cancer immunotherapy. Nevertheless, the roles of DUBs in the crosstalk between tumors and their surrounding components have not been comprehensively reviewed. In this review, we discuss the involvement of DUBs in the dynamic interplay between tumors, immune cells, and stromal cells and illustrate how dysregulated DUBs facilitate immune evasion and promote tumor progression. We also summarize potential small molecules that target DUBs to alleviate immunosuppression and suppress tumorigenesis. Finally, we discuss the prospects and challenges regarding the targeting of DUBs in cancer immunotherapeutics and several urgent problems that warrant further investigation.


Assuntos
Enzimas Desubiquitinantes , Microambiente Tumoral , Humanos , Microambiente Tumoral/imunologia , Enzimas Desubiquitinantes/metabolismo , Animais , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/enzimologia , Neoplasias/metabolismo , Evasão Tumoral , Ubiquitinação , Evasão da Resposta Imune
2.
Chem Biol Drug Des ; 103(5): e14530, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38725091

RESUMO

Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.


Assuntos
Transcriptase Reversa do HIV , Vírus da Imunodeficiência Felina , Inibidores da Transcriptase Reversa , Animais , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/química , Gatos , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , Humanos , Relação Estrutura-Atividade , Pirimidinas/química , Pirimidinas/farmacologia , Alcinos/química , Alcinos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Ciclopropanos/farmacologia , Ciclopropanos/química , Simulação de Acoplamento Molecular , Benzoxazinas/química , Benzoxazinas/farmacologia
3.
Open Biol ; 14(5): 240014, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38745462

RESUMO

Most successes in computational protein engineering to date have focused on enhancing one biophysical trait, while multi-trait optimization remains a challenge. Different biophysical properties are often conflicting, as mutations that improve one tend to worsen the others. In this study, we explored the potential of an automated computational design strategy, called CamSol Combination, to optimize solubility and stability of enzymes without affecting their activity. Specifically, we focus on Bacillus licheniformis α-amylase (BLA), a hyper-stable enzyme that finds diverse application in industry and biotechnology. We validate the computational predictions by producing 10 BLA variants, including the wild-type (WT) and three designed models harbouring between 6 and 8 mutations each. Our results show that all three models have substantially improved relative solubility over the WT, unaffected catalytic rate and retained hyper-stability, supporting the algorithm's capacity to optimize enzymes. High stability and solubility embody enzymes with superior resilience to chemical and physical stresses, enhance manufacturability and allow for high-concentration formulations characterized by extended shelf lives. This ability to readily optimize solubility and stability of enzymes will enable the rapid and reliable generation of highly robust and versatile reagents, poised to contribute to advancements in diverse scientific and industrial domains.


Assuntos
Proteínas de Bactérias , Estabilidade Enzimática , Engenharia de Proteínas , Solubilidade , alfa-Amilases , alfa-Amilases/química , alfa-Amilases/metabolismo , alfa-Amilases/genética , Engenharia de Proteínas/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mutação , Bacillus licheniformis/enzimologia , Bacillus licheniformis/genética , Algoritmos , Modelos Moleculares
4.
Biol Lett ; 20(5): 20230585, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38746983

RESUMO

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.


Assuntos
Crustáceos , Sulfotransferases , Animais , Sulfotransferases/metabolismo , Sulfotransferases/genética , Crustáceos/enzimologia , Crustáceos/genética , Crustáceos/metabolismo , Filogenia , Evolução Molecular , Luminescência
5.
Pak J Pharm Sci ; 37(1(Special)): 199-203, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38747270

RESUMO

This study investigates the combined effect of vitamin C and chromium on BMI, lipid profile, LFTs and HbA1c of Diabetes Mellitus type 2 patients. This is randomized controlled trial study. For this study a total of 60 patients (n=28 female, n=32 male) Diabetes Mellitus type 2 patients were selected. They were divided into treatment group (vitamin C (500mg) Chromium (200µg) and control group (placebo) comprising thirty patients per group. Mean age in control group and treatment group is 33± 5.729 and 33±7.017 respectively. Statistical analysis showed significant results of lipid profile; total cholesterol (mg/dl) 198±66.1 P=0.008, High-Density Lipoprotein 38±7.5, P<0.001, Low Density Lipoprotein (LDL) (mg/dl) 105.1±22.4, P=0.002 and Triglycerides 191±64.3, P=0.02 are respectively. Levels of serum ALT (u/l) (34.7±9.1, P<0.001) and AST (u/l) (31.6 ±8.6, P<0.001) were significantly lower as compared to control group. HbA1c percentages were also normalized (5.45±0.2, P<.001) as compared to group 2. BMI values were also improved (P=0.01) after treatment. Combined supplementation of vitamin C and chromium reduce the plasma lipid percentage, blood glucose levels and also improve the ALT and AST functions.


Assuntos
Ácido Ascórbico , Índice de Massa Corporal , Cromo , Diabetes Mellitus Tipo 2 , Hemoglobinas Glicadas , Humanos , Feminino , Masculino , Ácido Ascórbico/uso terapêutico , Cromo/uso terapêutico , Adulto , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hemoglobinas Glicadas/metabolismo , Hiperglicemia/tratamento farmacológico , Hiperglicemia/sangue , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/sangue , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Pessoa de Meia-Idade
6.
Braz Oral Res ; 38: e034, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38747821

RESUMO

The aim of this study was to investigate the effect of acid challenge on the activation of matrix metalloproteinases (MMPs) in the Dentinoenamel junction of primary and permanent teeth submitted to radiotherapy. For this purpose, a total of 178 dental fragments obtained from molars were used, and randomly divided into 2 groups (primary and permanent teeth) / 4 experimental subgroups (irradiated and non-irradiated, demineralized and non-demineralized). The fragments were exposed to radiation, with a dose fraction of 2 Gy, for 5 consecutive days, until a total dose of 60 Gy was reached, with a total of 30 cycles, for 6 weeks. To determine the activity of MMPs on the dentinoenamel junction (DEJ), in situ zymography assays on 0.6mm dental fragments were performed. To assess whether MMP activity would be impacted by an acidic environment, the fragments were placed in a demineralizing solution (pH of 4.8). The finding was that irradiation activated MMPs in DEJ and these effects were more evident in permanent when compared with primary teeth. When the effect of an acid challenge on MMPs activity was investigated, demineralization was observed not to increase MMPs activity in non-irradiated teeth, but it did increase MMPs activity in irradiated teeth. In conclusion, an acid challenge was found to exacerbate activation of MMPs in DEJ of permanent teeth submitted to irradiation, but not in primary teeth.


Assuntos
Metaloproteinases da Matriz , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz/efeitos da radiação , Metaloproteinases da Matriz/análise , Humanos , Fatores de Tempo , Dente Decíduo/efeitos da radiação , Dente Decíduo/efeitos dos fármacos , Dentina/efeitos da radiação , Dentina/efeitos dos fármacos , Dentina/enzimologia , Dentição Permanente , Distribuição Aleatória , Concentração de Íons de Hidrogênio , Desmineralização do Dente , Estatísticas não Paramétricas , Análise de Variância , Valores de Referência , Ativação Enzimática/efeitos da radiação , Ativação Enzimática/efeitos dos fármacos
7.
Molecules ; 29(9)2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38731401

RESUMO

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.


Assuntos
Azadirachta , Di-Hidro-Orotato Desidrogenase , Simulação de Acoplamento Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Esquistossomose , Azadirachta/química , Animais , Esquistossomose/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Humanos , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/química , Simulação de Dinâmica Molecular , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/enzimologia , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Simulação por Computador , Esquistossomicidas/farmacologia , Esquistossomicidas/química , Esquistossomicidas/uso terapêutico , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Praziquantel/farmacologia , Praziquantel/química , Praziquantel/uso terapêutico
8.
Molecules ; 29(9)2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38731473

RESUMO

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.


Assuntos
Família Multigênica , Peptídeo Sintases , Policetídeo Sintases , Streptomyces , Streptomyces/genética , Streptomyces/enzimologia , Streptomyces/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeo Sintases/química , Peptídeo Sintases/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química
9.
Molecules ; 29(9)2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38731613

RESUMO

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 µM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors.


Assuntos
Fármacos Anti-HIV , Simulação de Acoplamento Molecular , Pirimidinas , Relação Quantitativa Estrutura-Atividade , Pirimidinas/química , Pirimidinas/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/síntese química , Humanos , Simulação de Dinâmica Molecular , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Estrutura Molecular
10.
Appl Microbiol Biotechnol ; 108(1): 332, 2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38734756

RESUMO

Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: • Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. • Three polyketides and one asterriquinone were isolated from HDAC deleted strain. • Two different PKSs were reported in C. olivaceum SD-80A.


Assuntos
Chaetomium , Histona Desacetilases , Família Multigênica , Policetídeos , Metabolismo Secundário , Chaetomium/genética , Chaetomium/enzimologia , Chaetomium/metabolismo , Metabolismo Secundário/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Policetídeos/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Vias Biossintéticas/genética , Epigênese Genética
11.
Appl Microbiol Biotechnol ; 108(1): 326, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38717487

RESUMO

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.


Assuntos
Escherichia coli , Streptomyces , Streptomyces/genética , Streptomyces/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Especificidade por Substrato , Dipeptidases/metabolismo , Dipeptidases/genética , Dipeptidases/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Família Multigênica , Concentração de Íons de Hidrogênio , Dipeptídeos/metabolismo , Temperatura , Cinética
12.
Food Res Int ; 186: 114364, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38729726

RESUMO

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.


Assuntos
Contaminação de Alimentos , Triticum , Zearalenona , Zearalenona/análise , Triticum/química , Triticum/microbiologia , Contaminação de Alimentos/análise , Bacillus megaterium/enzimologia , Descontaminação/métodos , Microbiologia de Alimentos , Manipulação de Alimentos/métodos , Bacillus/enzimologia , Sementes/química , Sementes/microbiologia , Microscopia Eletrônica de Varredura
13.
Sci Adv ; 10(19): eadk7283, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38728392

RESUMO

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.


Assuntos
Anidrases Carbônicas , Cianobactérias , Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/metabolismo , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Anidrases Carbônicas/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/química , Cianobactérias/metabolismo , Cianobactérias/genética , Cianobactérias/enzimologia , Regulação Alostérica , Filogenia , Ribulosefosfatos/metabolismo , Modelos Moleculares , Multimerização Proteica , Dióxido de Carbono/metabolismo , Especificidade por Substrato , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química
14.
J Am Chem Soc ; 146(19): 13399-13405, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38698691

RESUMO

Structural motifs containing nitrogen-nitrogen (N-N) bonds are prevalent in a large number of clinical drugs and bioactive natural products. Hydrazine (N2H4) serves as a widely utilized building block for the preparation of these N-N-containing molecules in organic synthesis. Despite its common use in chemical processes, no enzyme has been identified to catalyze the incorporation of free hydrazine in natural product biosynthesis. Here, we report that a hydrazine transferase catalyzes the condensation of N2H4 and an aromatic polyketide pathway intermediate, leading to the formation of a rare N-aminolactam pharmacophore in the biosynthesis of broad-spectrum antibiotic albofungin. These results expand the current knowledge on the biosynthetic mechanism for natural products with N-N units and should facilitate future development of biocatalysts for the production of N-N-containing chemicals.


Assuntos
Hidrazinas , Hidrazinas/química , Hidrazinas/metabolismo , Antibacterianos/química , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Streptomyces/enzimologia , Streptomyces/metabolismo , Lactamas/química , Lactamas/metabolismo , Farmacóforo
15.
J Agric Food Chem ; 72(19): 11002-11012, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38700031

RESUMO

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.


Assuntos
Biocatálise , Catecol O-Metiltransferase , Flavonoides , Proteínas Fúngicas , Cogumelos Shiitake , Cogumelos Shiitake/enzimologia , Cogumelos Shiitake/genética , Cogumelos Shiitake/química , Cogumelos Shiitake/metabolismo , Catecol O-Metiltransferase/genética , Catecol O-Metiltransferase/metabolismo , Catecol O-Metiltransferase/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Flavonoides/química , Flavonoides/metabolismo , Aromatizantes/metabolismo , Aromatizantes/química , Micélio/enzimologia , Micélio/genética , Micélio/química , Micélio/metabolismo , Especificidade por Substrato
16.
J Agric Food Chem ; 72(19): 11041-11050, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38700846

RESUMO

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g., cellulases and chitinases. Similar tunnel architecture is also observed in the Limosilactobacillus reuteri 121 GtfB (Lr121 GtfB) α-glucanotransferase from the GH70 family. The molecular element underpinning processivity of these transglucosylases remains underexplored. Here, we report the synthesis of the smallest (α1 → 4)-α-glucan interspersed with linear and branched (α1 → 6) linkages by a novel 4,6-α-glucanotransferase from L. reuteri N1 (LrN1 GtfB) with an open-clefted active center instead of the tunnel structure. Notably, the loop swapping engineering of LrN1 GtfB and Lr121 GtfB based on their crystal structures clarified the impact of the loop-mediated tunnel/cleft structure at the donor subsites -2 to -3 on processivity of these α-glucanotransferases, enabling the tailoring of both product sizes and substrate preferences. This study provides unprecedented insights into the processivity determinants and evolutionary diversification of GH70 α-glucanotransferases and offers a simple route for engineering starch-converting α-glucanotransferases to generate diverse α-glucans for different biotechnological applications.


Assuntos
Proteínas de Bactérias , Glucanos , Limosilactobacillus reuteri , Glucanos/química , Glucanos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Limosilactobacillus reuteri/enzimologia , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/química , Domínio Catalítico , Glucosiltransferases/química , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Engenharia de Proteínas , Sistema da Enzima Desramificadora do Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/química
17.
J Agric Food Chem ; 72(19): 10995-11001, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38701424

RESUMO

The titer of the microbial fermentation products can be increased by enzyme engineering. l-Sorbosone dehydrogenase (SNDH) is a key enzyme in the production of 2-keto-l-gulonic acid (2-KLG), which is the precursor of vitamin C. Enhancing the activity of SNDH may have a positive impact on 2-KLG production. In this study, a computer-aided semirational design of SNDH was conducted. Based on the analysis of SNDH's substrate pocket and multiple sequence alignment, three modification strategies were established: (1) expanding the entrance of SNDH's substrate pocket, (2) engineering the residues within the substrate pocket, and (3) enhancing the electron transfer of SNDH. Finally, mutants S453A, L460V, and E471D were obtained, whose specific activity was increased by 20, 100, and 10%, respectively. In addition, the ability of Gluconobacter oxidans WSH-004 to synthesize 2-KLG was improved by eliminating H2O2. This study provides mutant enzymes and metabolic engineering strategies for the microbial-fermentation-based production of 2-KLG.


Assuntos
Proteínas de Bactérias , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Gluconobacter/enzimologia , Gluconobacter/genética , Gluconobacter/metabolismo , Açúcares Ácidos/metabolismo , Açúcares Ácidos/química , Fermentação , Engenharia de Proteínas , Engenharia Metabólica , Desidrogenases de Carboidrato/metabolismo , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/química , Cinética
18.
J Agric Food Chem ; 72(19): 10772-10780, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38703122

RESUMO

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Herbicidas , Nicotiana , Proteínas de Plantas , Protoporfirinogênio Oxidase , Piridazinas , Protoporfirinogênio Oxidase/antagonistas & inibidores , Protoporfirinogênio Oxidase/metabolismo , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/genética , Piridazinas/química , Piridazinas/farmacologia , Herbicidas/farmacologia , Herbicidas/química , Herbicidas/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Nicotiana/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Simulação de Acoplamento Molecular , Estrutura Molecular , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Cinética
19.
J Agric Food Chem ; 72(19): 11221-11229, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38703356

RESUMO

Liposcelis bostrychophila, commonly known as booklouse, is an important stored-product pest worldwide. Studies have demonstrated that booklices have developed resistance to several insecticides. In this study, an integument esterase gene, LbEST-inte4, with upregulated expression, was characterized in L. bostrychophila. Knockdown of LbEST-inte4 resulted in a substantial increase in the booklice susceptibility to malathion. Overexpression of LbEST-inte4 in Drosophila melanogaster significantly enhanced its malathion tolerance. Molecular modeling and docking analysis suggested potential interactions between LbEST-inte4 and malathion. When overexpressed LbEST-inte4 in Sf9 cells, a notable elevation in esterase activity and malathion tolerance was observed. HPLC analysis indicated that the LbEST-inte4 enzyme could effectively degrade malathion. Taken together, the upregulated LbEST-inte4 appears to contribute to malathion tolerance in L. bostrychophila by facilitating the depletion of malathion. This study elucidates the molecular mechanism underlying malathion detoxification and provides the foundations for the development of effective prevention and control measures against psocids.


Assuntos
Esterases , Proteínas de Insetos , Insetos , Inseticidas , Malation , Animais , Malation/metabolismo , Malation/química , Malation/toxicidade , Malation/farmacologia , Inseticidas/metabolismo , Inseticidas/química , Inseticidas/farmacologia , Esterases/metabolismo , Esterases/genética , Esterases/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Insetos/efeitos dos fármacos , Resistência a Inseticidas/genética , Inativação Metabólica , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo
20.
Curr Microbiol ; 81(6): 163, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710822

RESUMO

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.


Assuntos
Integrases , Integrons , Recombinação Genética , Integrases/genética , Integrases/metabolismo , Integrons/genética , Mutação , Escherichia coli/genética , Escherichia coli/enzimologia , Bactérias/genética , Bactérias/enzimologia
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