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1.
J Inorg Biochem ; 260: 112688, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39111220

RESUMO

New-to-Nature biocatalysis has emerged as a promising tool in organic synthesis thanks to progress in protein engineering. Notably, hemeproteins have been evolved into robust catalysts for carbene and nitrene transfers and related sigmatropic rearrangements. In this work, we report the first example of a [2,3]-sigmatropic Sommelet-Hauser rearrangement initiated by a carbene transfer of the sperm whale myoglobin mutant L29S,H64V,V68F that was previously reported to catalyze the mechanistically similar [2,3]-sigmatropic Doyle-Kirmse rearrangement. This repurposed heme enzyme catalyzes the Sommelet-Hauser rearrangement between ethyl diazoacetate and benzyl thioethers bearing strong electron-withdrawing substituents with good yields and enantiomeric excess. Optimized catalytic conditions in the absence of any reductant led to an increased asymmetric induction with up to 59% enantiomeric excess. This myoglobin mutant is therefore one of the few catalysts for the asymmetric Sommelet-Hauser rearrangement. This work broadens the scope of abiological reactions catalyzed by iron-carbene transferases with a new example of asymmetric sigmatropic rearrangement.


Assuntos
Mioglobina , Mioglobina/química , Mioglobina/genética , Mioglobina/metabolismo , Metano/análogos & derivados , Metano/química , Metano/metabolismo , Biocatálise , Transferases/metabolismo , Transferases/genética , Transferases/química , Animais , Cachalote , Engenharia de Proteínas/métodos
2.
Nat Commun ; 15(1): 6642, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-39103329

RESUMO

Plasmodium falciparum is the main causative agent of malaria, a deadly disease that mainly affects children under five years old. Artemisinin-based combination therapies have been pivotal in controlling the disease, but resistance has arisen in various regions, increasing the risk of treatment failure. The non-mevalonate pathway is essential for the isoprenoid synthesis in Plasmodium and provides several under-explored targets to be used in the discovery of new antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of the pathway. Despite its importance, there are no structures available for any Plasmodium spp., due to the complex sequence which contains large regions of high disorder, making crystallisation a difficult task. In this manuscript, we use cryo-electron microscopy to solve the P. falciparum DXPS structure at a final resolution of 2.42 Å. Overall, the structure resembles other DXPS enzymes but includes a distinct N-terminal domain exclusive to the Plasmodium genus. Mutational studies show that destabilization of the cap domain interface negatively impacts protein stability and activity. Additionally, a density for the co-factor thiamine diphosphate is found in the active site. Our work highlights the potential of cryo-EM to obtain structures of P. falciparum proteins that are unfeasible by means of crystallography.


Assuntos
Microscopia Crioeletrônica , Plasmodium falciparum , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Pentosiltransferases/metabolismo , Pentosiltransferases/química , Pentosiltransferases/genética , Pentosiltransferases/ultraestrutura , Domínios Proteicos , Modelos Moleculares , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , Transferases
3.
Sci Rep ; 14(1): 18930, 2024 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-39147804

RESUMO

VQ1 and VQ10 are largely unstructured homologous proteins with a significant potential for protein-protein interactions. Yeast two-hybrid (Y2H) analysis confirmed that both proteins interact not only with themselves and each other but also with other VQ and WRKY proteins. Screening an Arabidopsis Y2H library with VQ1 as bait identified 287 interacting proteins. Validation of the screening confirmed that interactions with VQ1 also occurred with VQ10, supporting their functional homology. Although VQ1 or VQ10 proteins do not localize in plastids, 47 VQ1-targets were found to be plastidial proteins. In planta interaction with the isoprenoid biosynthetic enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was confirmed by co-immunoprecipitation. DXS oligomerizes through redox-regulated intermolecular disulfide bond formation, and the interaction with VQ1 or VQ10 do not involve their unique C residues. The VQ-DXS protein interaction did not alter plastid DXS localization or its oligomerization state. Although plants with enhanced or reduced VQ1 and VQ10 expression did not exhibit significantly altered levels of isoprenoids compared to wild-type plants, they did display significantly improved or diminished photosynthesis efficiency, respectively.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Plastídeos , Transferases , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Plastídeos/metabolismo , Transferases/metabolismo , Transferases/genética , Técnicas do Sistema de Duplo-Híbrido , Ligação Proteica , Motivos de Aminoácidos , Regulação da Expressão Gênica de Plantas
4.
Mar Drugs ; 22(7)2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-39057401

RESUMO

Four tunicamycin class compounds, tunicamycin VII (1), tunicamycin VIII (2), corynetoxin U17a (3), and tunicamycin IX (4), were isolated from the culture broth of the marine-derived actinomycete Streptomyces sp. MBTG32. The strain was identified using the 16S rDNA sequencing technique, and the isolated strain was closely related to Streptomyces bacillaris. The structures of the isolated compounds were elucidated based on spectroscopic data and comparisons with previously reported NMR data. Compounds 1-4 showed potent antibacterial activities against Gram-positive bacteria, especially Staphylococcus aureus, with MIC values of 0.13-0.25 µg/mL. Through a recombinant enzyme assay and overexpression analysis, we found that the isolated compounds exerted potent inhibitory effects on S. aureus MurNAc-pentapeptide translocase (MraY), with IC50 values of 0.08-0.21 µg/mL. The present results support that the underlying mechanism of action of tunicamycins isolated from marine-derived Streptomyces sp. is also associated with the inhibition of MraY enzyme activity in S. aureus.


Assuntos
Antibacterianos , Proteínas de Bactérias , Testes de Sensibilidade Microbiana , Staphylococcus aureus , Streptomyces , Tunicamicina , Staphylococcus aureus/efeitos dos fármacos , Tunicamicina/farmacologia , Antibacterianos/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos) , Transferases/antagonistas & inibidores , Transferases/metabolismo , Organismos Aquáticos
5.
N Biotechnol ; 83: 66-73, 2024 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-38960021

RESUMO

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis.


Assuntos
Dimetilaliltranstransferase , Diterpenos , Diterpenos/metabolismo , Dimetilaliltranstransferase/metabolismo , Dimetilaliltranstransferase/genética , Pentosiltransferases/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/biossíntese , Alcenos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Transferases
6.
Ann Clin Transl Neurol ; 11(6): 1615-1629, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38750253

RESUMO

OBJECTIVE: COASY, the gene encoding the bifunctional enzyme CoA synthase, which catalyzes the last two reactions of cellular de novo coenzyme A (CoA) biosynthesis, has been linked to two exceedingly rare autosomal recessive disorders, such as COASY protein-associated neurodegeneration (CoPAN), a form of neurodegeneration with brain iron accumulation (NBIA), and pontocerebellar hypoplasia type 12 (PCH12). We aimed to expand the phenotypic spectrum and gain insights into the pathogenesis of COASY-related disorders. METHODS: Patients were identified through targeted or exome sequencing. To unravel the molecular mechanisms of disease, RNA sequencing, bioenergetic analysis, and quantification of critical proteins were performed on fibroblasts. RESULTS: We identified five new individuals harboring novel COASY variants. While one case exhibited classical CoPAN features, the others displayed atypical symptoms such as deafness, language and autism spectrum disorders, brain atrophy, and microcephaly. All patients experienced epilepsy, highlighting its potential frequency in COASY-related disorders. Fibroblast transcriptomic profiling unveiled dysregulated expression in genes associated with mitochondrial respiration, responses to oxidative stress, transmembrane transport, various cellular signaling pathways, and protein translation, modification, and trafficking. Bioenergetic analysis revealed impaired mitochondrial oxygen consumption in COASY fibroblasts. Despite comparable total CoA levels to control cells, the amounts of mitochondrial 4'-phosphopantetheinylated proteins were significantly reduced in COASY patients. INTERPRETATION: These results not only extend the clinical phenotype associated with COASY variants but also suggest a continuum between CoPAN and PCH12. The intricate interplay of altered cellular processes and signaling pathways provides valuable insights for further research into the pathogenesis of COASY-associated diseases.


Assuntos
Fenótipo , Transcriptoma , Humanos , Masculino , Feminino , Criança , Pré-Escolar , Epilepsia/genética , Fibroblastos/metabolismo , Adolescente , Transtorno do Espectro Autista/genética , Adulto , Transferases
7.
Biochem Biophys Res Commun ; 722: 150160, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-38795453

RESUMO

Carbene transfer reactions have emerged as pivotal methodologies for the synthesis of complex molecular architectures. Heme protein-catalyzed carbene transfer reactions have shown promising results on model compounds. However, their limited substrate scope has hindered their application in natural product functionalization. Building upon the foundation of previously published work on a carbene transferase-myoglobin variant, this study employs computer-aided protein engineering to design myoglobin variants, using either docking or the deep learning-based LigandMPNN method. These variants were utilized as catalysts in carbene transfer reactions with a selection of monoterpene substrates featuring C-C double bonds, leading to seven target products. This cost-effective methodology broadens the substrate scope for heme protein-catalyzed reactions, thereby opening novel pathways for research in heme protein functionalities and offering fresh perspectives in the synthesis of bioactive molecules.


Assuntos
Metano , Monoterpenos , Mioglobina , Mioglobina/química , Metano/química , Metano/análogos & derivados , Monoterpenos/química , Monoterpenos/metabolismo , Engenharia de Proteínas/métodos , Transferases/química , Transferases/metabolismo , Simulação de Acoplamento Molecular
8.
Sci Rep ; 14(1): 8978, 2024 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-38637685

RESUMO

tRNA modifications play a crucial role in ensuring accurate codon recognition and optimizing translation levels. While the significance of these modifications in eukaryotic cells for maintaining cellular homeostasis and physiological functions is well-established, their physiological roles in bacterial cells, particularly in pathogenesis, remain relatively unexplored. The TusDCB protein complex, conserved in γ-proteobacteria like Escherichia coli, is involved in sulfur modification of specific tRNAs. This study focused on the role of TusDCB in the virulence of uropathogenic E. coli (UPEC), a bacterium causing urinary tract infections. The findings indicate that TusDCB is essential for optimal production of UPEC's virulence factors, including type 1 fimbriae and flagellum, impacting the bacterium's ability to aggregate in bladder epithelial cells. Deletion of tusDCB resulted in decreased virulence against urinary tract infection mice. Moreover, mutant TusDCB lacking sulfur transfer activity and tusE- and mnmA mutants revealed the indispensability of TusDCB's sulfur transfer activity for UPEC pathogenicity. The study extends its relevance to highly pathogenic, multidrug-resistant strains, where tusDCB deletion reduced virulence-associated bacterial aggregation. These insights not only deepen our understanding of the interplay between tRNA sulfur modification and bacterial pathogenesis but also highlight TusDCB as a potential therapeutic target against UPEC strains resistant to conventional antimicrobial agents.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Camundongos , Virulência/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/microbiologia , Fatores de Virulência/genética , Transferases/metabolismo
9.
Biochemistry (Mosc) ; 89(2): 241-256, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38622093

RESUMO

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.


Assuntos
Oxirredutases , Vibrio , Oxirredutases/metabolismo , NAD/metabolismo , Cinamatos , Oxirredução , Vibrio/genética , Vibrio/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADH Desidrogenase/metabolismo , Flavinas/química , Transferases , Flavina-Adenina Dinucleotídeo/metabolismo
10.
Int J Mol Sci ; 25(8)2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38673766

RESUMO

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.


Assuntos
Eritritol , Eritritol/análogos & derivados , Populus , Fosfatos Açúcares , Transferases , Populus/genética , Populus/metabolismo , Populus/enzimologia , Eritritol/metabolismo , Fosfatos Açúcares/metabolismo , Transferases/metabolismo , Transferases/genética , Hemiterpenos/metabolismo , Butadienos/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Pentanos/metabolismo , Plantas Geneticamente Modificadas
11.
Am J Med Genet A ; 194(8): e63622, 2024 08.
Artigo em Inglês | MEDLINE | ID: mdl-38572626

RESUMO

Nonketotic hyperglycinemia (NKH) is a relatively well-characterized inborn error of metabolism that results in a combination of lethargy, hypotonia, seizures, developmental arrest, and, in severe cases, death early in life. Three genes encoding components of the glycine cleavage enzyme system-GLDC, AMT, and GCSH-are independently associated with NKH. We report on a patient with severe NKH in whom the homozygous pathogenic variant in AMT (NM_000481.3):c.602_603del (p.Lys201Thrfs*75) and the homozygous likely pathogenic variant in GLDC(NM_000170.2):c.2852C>A (p.Ser951Tyr) were both identified. Our patient demonstrates a novel combination of two homozygous disease-causing variants impacting the glycine cleavage pathway at two different components, and elicits management- and genetic counseling-related challenges for the family.


Assuntos
Homozigoto , Hiperglicinemia não Cetótica , Humanos , Hiperglicinemia não Cetótica/genética , Hiperglicinemia não Cetótica/patologia , Masculino , Glicina Desidrogenase (Descarboxilante)/genética , Aminometiltransferase/genética , Feminino , Mutação/genética , Lactente , Glicina/genética , Recém-Nascido , Fenótipo , Predisposição Genética para Doença , Aminoácido Oxirredutases , Complexos Multienzimáticos , Transferases
12.
Sci Rep ; 14(1): 5765, 2024 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-38459140

RESUMO

Autism spectrum disorder (ASD) is a complicated, lifelong neurodevelopmental disorder affecting verbal and non-verbal communication and social interactions. ASD signs and symptoms appear early in development before the age of 3 years. It is unlikely for a person to acquire autism after a period of normal development. However, we encountered an 8-year-old child who developed ASD later in life although his developmental milestones were normal at the beginning of life. Sequencing the complete coding part of the genome identified a hemizygous nonsense mutation (NM_001367857.2):c.1803C>G; (p.Tyr601Ter) in the gene (SATL1) encoding spermidine/spermine N1-acetyl transferase like 1. Screening an ASD cohort of 28 isolated patients for the SATL1 gene identified another patient with the same variant. Although SATL1 mutations have not been associated with any human diseases, our data suggests that a mutation in SATL1 is the underlying cause of ASD in our cases. In mammals, mutations in spermine synthase (SMS), an enzyme needed for the synthesis of spermidine polyamine, have been reported in a syndromic form of the X-linked mental retardation. Moreover, SATL1 gene expression studies showed a relatively higher expression of SATL1 transcripts in ASD related parts of the brain including the cerebellum, amygdala and frontal cortex. Additionally, spermidine has been characterized in the context of learning and memory and supplementations with spermidine increase neuroprotective effects and decrease age-induced memory impairment. Furthermore, spermidine biosynthesis is required for spontaneous axonal regeneration and prevents α-synuclein neurotoxicity in invertebrate models. Thus, we report, for the first time, that a mutation in the SATL1 gene could be a contributing factor in the development of autistic symptoms in our patients.


Assuntos
Transtorno do Espectro Autista , Espermidina , Animais , Criança , Humanos , Transtorno do Espectro Autista/genética , Poliaminas/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Transferases
13.
Carbohydr Res ; 538: 109095, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38507941

RESUMO

Moraxella nonliquefaciens is a commensal of the human upper respiratory tract (URT) but on rare occasions is recovered in cases of ocular, septic and pulmonary infections. Hence there is interest in the pathogenic determinants of M. nonliquefaciens, of which outer membrane (OM) structures such as fimbriae and two capsular polysaccharide (CPS) structures, →3)-ß-D-GalpNAc-(1→5)-ß-Kdop-(2→ and →8)-α-NeuAc-(2→, have been reported in the literature. To further characterise its surface virulence factors, we isolated a novel CPS from M. nonliquefaciens type strain CCUG 348T. This structure was elucidated using NMR data obtained from CPS samples that were subjected to various degrees of mild acid hydrolysis. Together with GLC-MS data, the structure was resolved as a linear polymer composed of two GalfNAc residues consecutively added to Kdo, →3)-ß-D-GalfNAc-(1→3)-α-D-GalfNAc-(1→5)-α-(8-OAc)Kdop-(2→. Supporting evidence for this material being CPS was drawn from the proposed CPS biosynthetic locus which encoded a potential GalfNAc transferase, a UDP-GalpNAc mutase for UDP-GalfNAc production and a putative CPS polymerase with predicted GalfNAc and Kdo transferase domains. This study describes a unique CPS composition reported in Moraxella spp. and offers genetic insights into the synthesis and expression of GalfNAc residues, which are rare in bacterial OM glycans.


Assuntos
Moraxella , Polissacarídeos , Humanos , Polissacarídeos/análise , Transferases/análise , Difosfato de Uridina/análise , Cápsulas Bacterianas/química , Polissacarídeos Bacterianos/química
14.
Plant Physiol Biochem ; 208: 108506, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38461753

RESUMO

Acetolactate synthase inhibitors (ALS inhibitors) and glyphosate are two classes of herbicides that act by inhibiting an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. Besides amino acid synthesis inhibition, both herbicides trigger similar physiological effects in plants. The main aim of this study was to evaluate the role of glutathione metabolism, with special emphasis on glutathione S-transferases (GSTs), in the mode of action of glyphosate and ALS inhibitors in Amaranthus palmeri. For that purpose, plants belonging to a glyphosate-sensitive (GLS) and a glyphosate-resistant (GLR) population were treated with different doses of glyphosate, and plants belonging to an ALS-inhibitor sensitive (AIS) and an ALS-inhibitor resistant (AIR) population were treated with different doses of the ALS inhibitor nicosulfuron. Glutathione-related contents, GST activity, and related gene expressions (glutamate-cysteine ligase, glutathione reductase, Phi GST and Tau GST) were analysed in leaves. According to the results of the analytical determinations, there were virtually no basal differences between GLS and GLR plants or between AIS and AIR plants. Glutathione synthesis and turnover did not follow a clear pattern in response to herbicides, but GST activity and gene expression (especially Phi GSTs) increased with both herbicides in treated sensitive plants, possibly related to the rocketing H2O2 accumulation. As GSTs offered the clearest results, these were further investigated with a multiple resistant (MR) population, compressing target-site resistance to both glyphosate and the ALS inhibitor pyrithiobac. As in single-resistant plants, measured parameters in the MR population were unaffected by herbicides, meaning that the increase in GST activity and expression occurs due to herbicide interactions with the target enzymes.


Assuntos
Amaranthus , Herbicidas , Herbicidas/farmacologia , Herbicidas/metabolismo , Peróxido de Hidrogênio/metabolismo , Resistência a Herbicidas , Glifosato , Glutationa/metabolismo , Transferases/metabolismo
15.
Int J Mol Sci ; 25(5)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38473957

RESUMO

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.


Assuntos
Acetatos , Carthamus tinctorius , Ciclopentanos , Oxilipinas , Transferases , Transferases/metabolismo , Ácido Clorogênico/metabolismo , Carthamus tinctorius/genética , Simulação de Acoplamento Molecular , Transcriptoma , Nucleotidiltransferases/metabolismo , Regulação da Expressão Gênica de Plantas
16.
Antonie Van Leeuwenhoek ; 117(1): 47, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38427176

RESUMO

Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17T. Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17T, lacks several genes from the methyl transferase operon found in strain 17T. The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol.


Assuntos
Álcool Desidrogenase , Metanol , Peptococcaceae , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Metanol/metabolismo , Oxirredução , Transferases/metabolismo , Sulfatos/metabolismo , Cobalto , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo
17.
Redox Biol ; 71: 103094, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38479221

RESUMO

Low-molecular-weight (LMW) thiols are produced in all living cells in different forms and concentrations. Glutathione (GSH), coenzyme A (CoA), bacillithiol (BSH), mycothiol (MSH), ergothioneine (ET) and trypanothione T(SH)2 are the main LMW thiols in eukaryotes and prokaryotes. LMW thiols serve as electron donors for thiol-dependent enzymes in redox-mediated metabolic and signaling processes, protect cellular macromolecules from oxidative and xenobiotic stress, and participate in the reduction of oxidative modifications. The level and function of LMW thiols, their oxidized disulfides and mixed disulfide conjugates in cells and tissues is tightly controlled by dedicated oxidoreductases, such as peroxiredoxins, glutaredoxins, disulfide reductases and LMW thiol transferases. This review provides the first summary of the current knowledge of structural and functional diversity of transferases for LMW thiols, including GSH, BSH, MSH and T(SH)2. Their role in maintaining redox homeostasis in single-cell and multicellular organisms is discussed, focusing in particular on the conjugation of specific thiols to exogenous and endogenous electrophiles, or oxidized protein substrates. Advances in the development of new research tools, analytical methodologies, and genetic models for the analysis of known LMW thiol transferases will expand our knowledge and understanding of their function in cell growth and survival under oxidative stress, nutrient deprivation, and during the detoxification of xenobiotics and harmful metabolites. The antioxidant function of CoA has been recently discovered and the breakthrough in defining the identity and functional characteristics of CoA S-transferase(s) is soon expected.


Assuntos
Antioxidantes , Compostos de Sulfidrila , Compostos de Sulfidrila/metabolismo , Antioxidantes/metabolismo , Transferases/metabolismo , Oxirredução , Glutationa/metabolismo , Oxirredutases/metabolismo , Dissulfetos/química
18.
Clin Neurol Neurosurg ; 239: 108189, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38437773

RESUMO

BACKGROUND: Levodopa treatment requires the addition of other drugs, such as catechol-O-methyl transferase (COMT) inhibitors, to alleviate motor fluctuations in advanced parkinson's disease (PD). However, the optimal strategy, including the type and dose of COMT inhibitors remains unknown. This systematic review and network meta-analysis aimed to assess the efficacy and safety of different COMT inhibitors and for treating PD patients. METHODS: PubMed, Embase, Cochrane Library and Web of Science were screened up to November 20, 2022. Randomized controlled trials (RCTs) of COMT inhibitors (entacapone, opicapone, tolcapone) for PD patients were included. Eligible outcomes were total ON-time, rate of ON-time >1 h, total daily dose of levodopa therapy, mean change from baseline to final follow up in Unified Parkinson's Disease Rating Scale (UPDRS) part III scores, adverse events and dyskinesia. Network meta-analyses integrated direct and indirect evidence with placebo as a common comparator. RESULTS: We identified 18 studies with 7564 patients. Opicapone, entacapone, and tolcapone could increase total ON-time when compared with placebo. However, opicapone (25 mg, MD 4.0, 95%CrI: 1.1-7.5) and opicapone (50 mg, MD 5.1, 95%CrI: 2.2-8.7) statistically significant increase the total ON-time. opicapone and entacapone could increase the rate of ON-time >1 h when compared with placebo. Only opicapone (5 mg) showed no statistically significant with placebo (OR 1.4, 95%CrI: 0.74-2.4). We found that opicapone (50 mg, SURCA, 0.796) is the best option compared with other treatments. TOL (200 mg) was ranked highest in the rank probability test for total daily dose of levodopa therapy, followed by OPI (50 mg), TOL (400 mg) and TOL (100 mg) in order. SUCRA rankings identified TOL (200 mg) as the most likely therapy for increasing adverse events (SUCRA 27.19%), followed by TOL (400 mg, SUCRA 27.20%) and OPI (5 mg, SUCRA 30.81%). The SUCRA probabilities were 91.6%, 75.2%, 67.9%, 59.3%, 45.6%, 41.1%, 35.1%, 24.6% and 9.4% for PLA, TOL (400 mg), ENT (100 mg), ENT (200 mg), OPI (5 mg), TOL (100 mg), OPI (25 mg), OPI (50 mg), and TOL (200 mg) respectively. CONCLUSION: In conclusion, opicapone (50 mg) may be a better choice for treatment PD when compared with other COMT inhibitors.


Assuntos
Nitrilas , Doença de Parkinson , Humanos , Doença de Parkinson/tratamento farmacológico , Levodopa/efeitos adversos , Antiparkinsonianos/efeitos adversos , Tolcapona/uso terapêutico , Metanálise em Rede , Inibidores de Catecol O-Metiltransferase/uso terapêutico , Inibidores de Catecol O-Metiltransferase/farmacologia , Catecóis/efeitos adversos , Transferases/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto
19.
ACS Infect Dis ; 10(4): 1312-1326, 2024 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-38513073

RESUMO

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)-based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure-activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.


Assuntos
Acetaldeído/análogos & derivados , Bactérias , Escherichia coli , Transferases , Antibacterianos/química , Piruvatos/metabolismo
20.
J Biol Chem ; 300(3): 105768, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38367664

RESUMO

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.


Assuntos
Galactanos , Mycobacterium , Galactanos/biossíntese , Polímeros/metabolismo , Proteômica , Transferases/metabolismo , Mycobacterium/metabolismo
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