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1.
Front Cell Infect Microbiol ; 14: 1413787, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38836053

RESUMO

Background: Trimethylamine-N-oxide (TMAO) is produced by hepatic flavin-containing monooxygenase 3 (FMO3) from trimethylamine (TMA). High TMAO level is a biomarker of cardiovascular diseases and metabolic disorders, and it also affects periodontitis through interactions with the gastrointestinal microbiome. While recent findings indicate that periodontitis may alter systemic TMAO levels, the specific mechanisms linking these changes and particular oral pathogens require further clarification. Methods: In this study, we established a C57BL/6J male mouse model by orally administering Porphyromonas gingivalis (P. gingivalis, Pg), Fusobacterium nucleatum (F. nucleatum, Fn), Streptococcus mutans (S. mutans, Sm) and PBS was used as a control. We conducted LC-MS/MS analysis to quantify the concentrations of TMAO and its precursors in the plasma and cecal contents of mice. The diversity and composition of the gut microbiome were analyzed using 16S rRNA sequencing. TMAO-related lipid metabolism and enzymes in the intestines and liver were assessed by qPCR and ELISA methods. We further explored the effect of Pg on FMO3 expression and lipid molecules in HepG2 cells by stimulating the cells with Pg-LPS in vitro. Results: The three oral pathogenic bacteria were orally administered to the mice for 5 weeks. The Pg group showed a marked increase in plasma TMAO, betaine, and creatinine levels, whereas no significant differences were observed in the gut TMAO level among the four groups. Further analysis showed similar diversity and composition in the gut microbiomes of both the Pg and Fn groups, which were different from the Sm and control groups. The profiles of TMA-TMAO pathway-related genera and gut enzymes were not significantly different among all groups. The Pg group showed significantly higher liver FMO3 levels and elevated lipid factors (IL-6, TG, TC, and NEFA) in contrast to the other groups. In vitro experiments confirmed that stimulation of HepG2 cells with Pg-LPS upregulated the expression of FMO3 and increased the lipid factors TC, TG, and IL-6. Conclusion: This study conclusively demonstrates that Pg, compared to Fn and Sm, plays a critical role in elevating plasma TMAO levels and significantly influences the TMA-TMAO pathway, primarily by modulating the expression of hepatic FMO3 and directly impacting hepatic lipid metabolism.


Assuntos
Microbioma Gastrointestinal , Metilaminas , Camundongos Endogâmicos C57BL , Oxigenases , Porphyromonas gingivalis , Animais , Masculino , Metilaminas/metabolismo , Metilaminas/sangue , Humanos , Camundongos , Oxigenases/metabolismo , Porphyromonas gingivalis/metabolismo , Fusobacterium nucleatum/metabolismo , Redes e Vias Metabólicas , Células Hep G2 , Metabolismo dos Lipídeos , Modelos Animais de Doenças , Periodontite/microbiologia , Periodontite/metabolismo , Fígado/metabolismo , RNA Ribossômico 16S/genética , Espectrometria de Massas em Tandem , Boca/microbiologia
2.
BMC Genomics ; 25(1): 469, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38745121

RESUMO

Carotenoid cleavage oxygenases (CCOs) enzymes play a vital role in plant growth and development through the synthesis of apocarotenoids and their derivative. These chemicals are necessary for flower and fruit coloration, as well as the manufacture of plant hormones such as abscisic acid (ABA) and strigolactones, which control a variety of physiological processes. The CCOs gene family has not been characterized in Arachis hypogaea. Genome mining of A. hypogaea identifies 24 AhCCO gene members. The AhCCO gene family was divided into two subgroups based on the recent study of the Arabidopsis thaliana CCO gene family classification system. Twenty-three AhCCO genes, constituting 95.8% of the total, were regulated by 29 miRNAs, underscoring the significance of microRNAs (miRNAs) in governing gene expression in peanuts. AhCCD19 is the only gene that lacks a miRNA target site. The physicochemical characteristics of CCO genes and their molecular weights and isoelectric points were studied further. The genes were then characterized regarding chromosomal distribution, structure, and promoter cis-elements. Light, stress development, drought stress, and hormone responsiveness were discovered to be associated with AhCCO genes, which can be utilized in developing more resilient crops. The investigation also showed the cellular location of the encoded proteins and discovered that the peanut carotenoid oxygenase gene family's expansion was most likely the result of tandem, segmental, and whole-genome duplication events. The localization expresses the abundance of genes mostly in the cytoplasm and chloroplast. Expression analysis shows that AhCCD7 and AhCCD14 genes show the maximum expression in the apical meristem, lateral leaf, and pentafoliate leaf development, while AhNCED9 and AhNCED13 express in response to Aspergillus flavus resistance. This knowledge throws light on the evolutionary history of the AhCCO gene family and may help researchers better understand the molecular processes behind gene duplication occurrences in plants. An integrated synteny study was used to find orthologous carotenoid oxygenase genes in A. hypogaea, whereas Arabidopsis thaliana and Beta vulgaris were used as references for the functional characterization of peanut CCO genes. These studies provide a foundation for future research on the regulation and functions of this gene family. This information provides valuable insights into the genetic regulation of AhCCO genes. This technology could create molecular markers for breeding programs to develop new peanut lines.


Assuntos
Arachis , Regulação da Expressão Gênica de Plantas , Família Multigênica , Oxigenases , Estresse Fisiológico , Arachis/genética , Arachis/enzimologia , Estresse Fisiológico/genética , Oxigenases/genética , Oxigenases/metabolismo , Carotenoides/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Filogenia , Genoma de Planta , Regiões Promotoras Genéticas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
3.
Plant Physiol Biochem ; 211: 108697, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38705045

RESUMO

Dunaliella salina, a microalga that thrives under high-saline conditions, is notable for its high ß-carotene content and the absence of a polysaccharide cell wall. These unique characteristics render it a prime candidate as a cellular platform for astaxanthin production. In this study, our initial tests in an E. coli revealed that ß-ring-4-dehydrogenase (CBFD) and 4-hydroxy-ß-ring-4-dehydrogenase (HBFD) genes from Adonis aestivalis outperformed ß-carotene hydroxylase (BCH) and ß-carotene ketolase (BKT) from Haematococcus pluvialis counterparts by two-fold in terms of astaxanthin biosynthesis efficiency. Subsequently, we utilized electroporation to integrate either the BKT gene or the CBFD and HBFD genes into the genome of D. salina. In comparison to wild-type D. salina, strains transformed with BKT or CBFD and HBFD exhibited inhibited growth, underwent color changes to shades of red and yellow, and saw a nearly 50% decline in cell density. HPLC analysis confirmed astaxanthin synthesis in engineered D. salina strains, with CBFD + HBFD-D. salina yielding 134.88 ± 9.12 µg/g of dry cell weight (DCW), significantly higher than BKT-D. salina (83.58 ± 2.40 µg/g). This represents the largest amount of astaxanthin extracted from transgenic D. salina, as reported to date. These findings have significant implications, opening up new avenues for the development of specialized D. salina-based microcell factories for efficient astaxanthin production.


Assuntos
Xantofilas , Xantofilas/metabolismo , Clorofíceas/metabolismo , Clorofíceas/genética , Vias Biossintéticas/genética , Clorófitas/metabolismo , Clorófitas/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Oxigenases de Função Mista , Oxigenases
4.
Nat Commun ; 15(1): 4399, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38782897

RESUMO

Soluble methane monooxygenase (sMMO) oxidizes a wide range of carbon feedstocks (C1 to C8) directly using intracellular NADH and is a useful means in developing green routes for industrial manufacturing of chemicals. However, the high-throughput biosynthesis of active recombinant sMMO and the ensuing catalytic oxidation have so far been unsuccessful due to the structural and functional complexity of sMMO, comprised of three functionally complementary components, which remains a major challenge for its industrial applications. Here we develop a catalytically active miniature of sMMO (mini-sMMO), with a turnover frequency of 0.32 s-1, through an optimal reassembly of minimal and modified components of sMMO on catalytically inert and stable apoferritin scaffold. We characterise the molecular characteristics in detail through in silico and experimental analyses and verifications. Notably, in-situ methanol production in a high-cell-density culture of mini-sMMO-expressing recombinant Escherichia coli resulted in higher yield and productivity (~ 3.0 g/L and 0.11 g/L/h, respectively) compared to traditional methanotrophic production.


Assuntos
Escherichia coli , Metanol , Oxigenases , Escherichia coli/genética , Escherichia coli/metabolismo , Oxigenases/metabolismo , Oxigenases/genética , Metanol/metabolismo , Metanol/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Oxirredução
5.
J Agric Food Chem ; 72(21): 12209-12218, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38751167

RESUMO

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.


Assuntos
Proteínas de Bactérias , Benzaldeídos , Ácidos Cumáricos , Oxigenases , Benzaldeídos/metabolismo , Benzaldeídos/química , Ácidos Cumáricos/metabolismo , Ácidos Cumáricos/química , Oxigenases/genética , Oxigenases/metabolismo , Oxigenases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Engenharia de Proteínas , Biocatálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Bacillus/enzimologia , Bacillus/genética
6.
Biotechnol Adv ; 73: 108374, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38729229

RESUMO

Indigo is a natural dye extensively used in the global textile industry. However, the conventional synthesis of indigo using toxic compounds like aniline, formaldehyde, and hydrogen cyanide has led to environmental pollution and health risks for workers. This method also faces growing economic, sustainability, and environmental challenges. To address these issues, the concept of bio-indigo or indigo biosynthesis has been proposed as an alternative to aniline-based indigo synthesis. Among various enzymes, Flavin-containing Monooxygenases (FMOs) have shown promise in achieving a high yield of bio-indigo. However, the industrialization of indigo biosynthesis still encounters several challenges. This review focuses on the historical development of indigo biosynthesis mediated by FMOs. It highlights several factors that have hindered industrialization, including the use of unsuitable chassis (Escherichia coli), the toxicity of indole, the high cost of the substrate L-tryptophan, the water-insolubility of the product indigo, the requirement of reducing reagents such as sodium dithionite, and the relatively low yield and high cost compared to chemical synthesis. Additionally, this paper summarizes various strategies to enhance the yield of indigo synthesized by FMOs, including redundant sequence deletion, semi-rational design, cheap precursor research, NADPH regeneration, large-scale fermentation, and enhancement of water solubility of indigo.


Assuntos
Índigo Carmim , Índigo Carmim/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases/metabolismo , Oxigenases/genética , Corantes/química , Corantes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo
7.
Sci Total Environ ; 934: 173046, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38735326

RESUMO

Although marine environments represent huge reservoirs of the potent greenhouse gas methane, they currently contribute little to global net methane emissions. Most of the methane is oxidized by methanotrophs, minimizing escape to the atmosphere. Aerobic methanotrophs oxidize methane mostly via the copper (Cu)-bearing enzyme particulate methane monooxygenase (pMMO). Therefore, aerobic methane oxidation depends on sufficient Cu acquisition by methanotrophs. Because they require both oxygen and methane, aerobic methanotrophs reside at oxic-anoxic interfaces, often close to sulphidic zones where Cu bioavailability can be limited by poorly soluble Cu sulphide mineral phases. Under Cu-limiting conditions, certain aerobic methanotrophs exude Cu-binding ligands termed chalkophores, such as methanobactin (mb) exuded by Methylosinus trichosporium OB3b. Our main objective was to establish whether chalkophores can mobilise Cu from Cu sulphide-bearing marine sediments to enhance Cu bioavailability. Through a series of kinetic batch experiments, we investigated Cu mobilisation by mb from a set of well-characterized sulphidic marine sediments differing in sediment properties, including Cu content and phase distribution. Characterization of solid-phase Cu speciation included X-ray absorption spectroscopy and a targeted sequential extraction. Furthermore, in batch experiments, we investigated to what extent adsorption of metal-free mb and Cu-mb complexes to marine sediments constrains Cu mobilisation. Our results are the first to show that both solid phase Cu speciation and chalkophore adsorption can constrain methanotrophic Cu acquisition from marine sediments. Only for certain sediments did mb addition enhance dissolved Cu concentrations. Cu mobilisation by mb was not correlated to the total Cu content of the sediment, but was controlled by solid-phase Cu speciation. Cu was only mobilised from sediments containing a mono-Cu-sulphide (CuSx) phase. We also show that mb adsorption to sediments limits Cu acquisition by mb to less compact (surface) sediments. Therefore, in sulphidic sediments, mb-mediated Cu acquisition is presumably constrained to surface-sediment interfaces containing mono-Cu-sulphide phases.


Assuntos
Cobre , Sedimentos Geológicos , Imidazóis , Methylosinus trichosporium , Oligopeptídeos , Cobre/metabolismo , Sedimentos Geológicos/química , Oligopeptídeos/metabolismo , Imidazóis/metabolismo , Imidazóis/química , Methylosinus trichosporium/metabolismo , Oxirredução , Metano/metabolismo , Oxigenases/metabolismo , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/análise
8.
Biochemistry ; 63(11): 1445-1459, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38779817

RESUMO

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.


Assuntos
Oxigenases de Função Mista , Óxidos de Nitrogênio , Oxirredução , Óxidos de Nitrogênio/metabolismo , Óxidos de Nitrogênio/química , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Cinética , Mutagênese Sítio-Dirigida , Flavinas/metabolismo , Flavinas/química , Modelos Moleculares , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Oxigenases
9.
PeerJ ; 12: e17337, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38784401

RESUMO

Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) stands as a widely cultivated leafy vegetable in China, with its leaf morphology significantly influencing both quality and yield. Despite its agricultural importance, the precise mechanisms governing leaf wrinkling development remain elusive. This investigation focuses on 'Wutacai', a representative cultivar of the Tacai variety (Brassica campestris L. ssp. chinensis var. rosularis Tsen et Lee), renowned for its distinct leaf wrinkling characteristics. Within the genome of 'Wutacai', we identified a total of 18 YUCs, designated as BraWTC_YUCs, revealing their conservation within the Brassica genus, and their close homology to YUCs in Arabidopsis. Expression profiling unveiled that BraWTC_YUCs in Chinese Cabbage exhibited organ-specific and leaf position-dependent variation. Additionally, transcriptome sequencing data from the flat leaf cultivar 'Suzhouqing' and the wrinkled leaf cultivar 'Wutacai' revealed differentially expressed genes (DEGs) related to auxin during the early phases of leaf development, particularly the YUC gene. In summary, this study successfully identified the YUC gene family in 'Wutacai' and elucidated its potential function in leaf wrinkling trait, to provide valuable insights into the prospective molecular mechanisms that regulate leaf wrinkling in Chinese cabbage.


Assuntos
Brassica , Regulação da Expressão Gênica de Plantas , Folhas de Planta , Brassica/genética , Brassica/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/anatomia & histologia , Perfilação da Expressão Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , China , Oxigenases/genética , Oxigenases/metabolismo , Genes de Plantas
10.
Nat Commun ; 15(1): 4226, 2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38762502

RESUMO

Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.


Assuntos
Metano , Óxido Nitroso , Óxido Nitroso/metabolismo , Metano/metabolismo , Concentração de Íons de Hidrogênio , Oxirredutases/metabolismo , Oxirredutases/genética , Oxigênio/metabolismo , Oxirredução , Anaerobiose , Metanol/metabolismo , Hidrogênio/metabolismo , Oxigenases/metabolismo , Oxigenases/genética
11.
J Biotechnol ; 389: 22-29, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38697360

RESUMO

Rieske non-heme iron oxygenases (ROs) are redox enzymes essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful biocatalysts due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H2O2) formation in the presence of a NADH cofactor regeneration system on the catalytic efficiency of CDO in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H2O2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h (TTN of 1515) at a 3-fold increase in production rate.


Assuntos
Peróxido de Hidrogênio , Oxigênio , Oxigênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxirredução , Oxigenases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , NAD/metabolismo , Sistema Livre de Células , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo
12.
Proc Natl Acad Sci U S A ; 121(16): e2311390121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38593075

RESUMO

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.


Assuntos
Archaea , Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Archaea/metabolismo , Fotossíntese , Glicolatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Oxigenases/metabolismo , Pentoses
13.
Physiol Plant ; 176(2): e14287, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38606719

RESUMO

Salt stress substantially leads to flowering delay. The regulation of salt-induced late flowering has been studied at the transcriptional and protein levels; however, the involvement of secondary metabolites has rarely been investigated. Here, we report that FMOGS-OXs (EC 1.14.13.237), the enzymes that catalyze the biosynthesis of glucosinolates (GSLs), promote flowering transition in Arabidopsis thaliana. It has been reported that WRKY75 is a positive regulator, and MAF4 is a negative regulator of flowering transition. The products of FMOGS-OXs, methylsulfinylalkyl GSLs (MS GSLs), facilitate flowering by inducing WRKY75 and repressing the MAS-MAF4 module. We further show that the degradation of MS GSLs is involved in salt-induced late flowering and salt tolerance. Salt stress induces the expression of myrosinase genes, resulting in the degradation of MS GSLs, thereby relieving the promotion of WRKY75 and inhibition of MAF4, leading to delayed flowering. In addition, the degradation products derived from MS GSLs enhance salt tolerance. Previous studies have revealed that FMOGS-OXs exhibit alternative catalytic activity to form trimethylamine N-oxide (TMAO) under salt stress, which activates multiple stress-related genes to promote salt tolerance. Therefore, FMOGS-OXs integrate flowering transition and salt tolerance in various ways. Our study shed light on the functional diversity of GSLs and established a connection between flowering transition, salt resistance, and GSL metabolism.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Oxigenases , Arabidopsis/metabolismo , Tolerância ao Sal , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosinolatos
14.
Molecules ; 29(7)2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38611907

RESUMO

The insecticidal property of ring C-seco limonoids has been discovered empirically and the target protein identified, but, to date, the molecular mechanism of action has not been described at the atomic scale. We elucidate on computational grounds whether nine C-seco limonoids present sufficiently high affinity to bind specifically with the putative target enzyme of the insects (ecdysone 20-monooxygenase). To this end, 3D models of ligands and the receptor target were generated and their interaction energies estimated by docking simulations. As a proof of concept, the tetrahydro-isoquinolinyl propenamide derivative QHC is the reference ligand bound to aldosterone synthase in the complex with PDB entry 4ZGX. It served as the 3D template for target modeling via homology. QHC was successfully docked back to its crystal pose in a one-digit nanomolar range. The reported experimental binding affinities span over the nanomolar to lower micromolar range. All nine limonoids were found with strong affinities in the range of -9 < ΔG < -13 kcal/mol. The molt hormone ecdysone showed a comparable ΔG energy of -12 kcal/mol, whereas -11 kcal/mol was the back docking result for the liganded crystal 4ZGX. In conclusion, the nine C-seco limonoids were strong binders on theoretical grounds in an activity range between a ten-fold lower to a ten-fold higher concentration level than insecticide ecdysone with its known target receptor. The comparable or even stronger binding hints at ecdysone 20-monooxygenase as their target biomolecule. Our assumption, however, is in need of future experimental confirmation before conclusions with certainty can be drawn about the true molecular mechanism of action for the C-seco limonoids under scrutiny.


Assuntos
Inseticidas , Limoninas , Oxigenases , Inseticidas/farmacologia , Ecdisona , Limoninas/farmacologia , Muda
15.
Sheng Wu Gong Cheng Xue Bao ; 40(4): 1076-1088, 2024 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-38658150

RESUMO

Flavin-containing monooxygenase (FMO) is the key enzyme in the biosynthesis pathway of CSOs with sulfur oxidation. In order to explore the molecular regulatory mechanism of FMO in the synthesis of onion CSOs, based on transcriptome database and phylogenetic analysis, one AcFMO gene that may be involved in alliin synthesis was obtained, the AcFMO had a cDNA of 1 374 bp and encoded 457 amino acids, which was evolutionarily closest to the AsFMO of garlic. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) indicated that AcFMO was the highest in the flowers and the lowest in the leaf sheaths. The results of subcellular localization showed that the AcFMO gene product was widely distributed throughout the cell A yeast expression vector was constructed, and the AcFMO gene was ecotopically overexpressed in yeast to further study the enzyme function in vitro and could catalyze the synthesis of alliin by S-allyl-l-cysteine. In summary, the cloning and functional identification of AcFMO have important reference value for understanding the biosynthesis of CSOs in onions.


Assuntos
Clonagem Molecular , Cisteína/análogos & derivados , Cebolas , Cebolas/genética , Cebolas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cisteína/biossíntese , Cisteína/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Sequência de Aminoácidos , Filogenia , Dissulfetos/metabolismo , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
16.
Biochemistry ; 63(9): 1170-1177, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38587906

RESUMO

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.


Assuntos
Imidazóis , Oligopeptídeos , Imidazóis/metabolismo , Imidazóis/química , Oligopeptídeos/metabolismo , Oligopeptídeos/química , Oligopeptídeos/biossíntese , Oxirredução , Cristalografia por Raios X , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Espectroscopia de Ressonância de Spin Eletrônica , Oxigenases/metabolismo , Oxigenases/química , Domínio Catalítico , Especificidade por Substrato , Modelos Moleculares , Ferro/metabolismo , Ferro/química
17.
Biochim Biophys Acta Mol Basis Dis ; 1870(5): 167188, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38657913

RESUMO

The incidence of gallbladder cholesterol stones (GCS) increases rapidly among people living in high-altitude hypoxic environments compared to those in normoxic areas. Upregulation of hepatic hypoxia inducible factor 1α (Hif-1α) plays a key role in the formation of GCS. High plasma trimethylamine-N-oxide (TMAO) levels are positively correlated with the occurrence of GCS. We hypothesized that HIF-1α may upregulate TMAO levels by promoting the transcription of flavin-containing monooxygenase 3 (Fmo3), which eventually leads to GCS formation. Our study shows that in women, high plasma total cholesterol and apolipoprotein B were positively correlated with cholecystolithiasis and hypoxia. Hif-1α binds to the Fmo3 promoter and promotes Fmo3 expression. Hypoxia and lithogenic diet induce the expression of Hif-1α, Fmo3, TMAO and cholesterol tube transporters in the livers of mice, disturb the proportion of bile and plasma components, and induce the formation of GCS. In cell experiments, silencing Hif-1α downregulates the expression of Fmo3, TMAO and cholesterol tube transporters. In a mouse model of hypoxic cholecystolithiasis, silencing Hif-1α downregulates the expression of related genes, restores the proportion of bile and plasma lipid components, and reduces the formation of GCS. Our study shows that Hif-1α binds to the promoter region of Fmo3 and promotes Fmo3 transcription. Thus, it mediates the transcriptional activation of the TMA/Fmo3/TMAO pathway, upregulates the expression of ATP-binding cassettes (Abc) g5 and g8, and participates in the regulation of the occurrence of GCS in the plateau region.


Assuntos
Colesterol , Cálculos Biliares , Subunidade alfa do Fator 1 Induzível por Hipóxia , Metilaminas , Oxigenases , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Animais , Humanos , Feminino , Camundongos , Colesterol/metabolismo , Cálculos Biliares/metabolismo , Cálculos Biliares/genética , Cálculos Biliares/patologia , Oxigenases/metabolismo , Oxigenases/genética , Metilaminas/metabolismo , Masculino , Vesícula Biliar/metabolismo , Vesícula Biliar/patologia , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Hipóxia/metabolismo , Hipóxia/genética , Adulto , Camundongos Endogâmicos C57BL , Colecistolitíase/metabolismo , Colecistolitíase/genética
18.
Molecules ; 29(6)2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38542906

RESUMO

(1) Background: Particulate methane monooxygenase (pMMO) has a strong dependence on the natural electron transfer path and is prone to denaturation, which results in its redox activity centers being unable to transfer electrons with bare electrodes directly and making it challenging to observe an electrochemical response; (2) Methods: Using methanobactin (Mb) as the electron transporter between gold electrodes and pMMO, a bionic interface with high biocompatibility and stability was created. The Mb-AuNPs-modified functionalized gold net electrode as a working electrode, the kinetic behaviors of pMMO bioelectrocatalysis, and the effect of Mb on pMMO were analyzed. The CV tests were performed at different scanning rates to obtain electrochemical kinetics parameters. (3) Results: The values of the electron transfer coefficient (α) and electron transfer rate constant (ks) are relatively large in test environments containing only CH4 or O2. In contrast, in the test environment containing both CH4 and O2, the bioelectrocatalysis of pMMO is a two-electron transfer process with a relatively small α and ks; (4) Conclusions: It was inferred that Mb formed the complex with pMMO. More importantly, Mb not only played a role in electron transfer but also in stabilizing the enzyme structure of pMMO and maintaining a specific redox state. Furthermore, the continuous catalytic oxidation of natural substrate methane was realized.


Assuntos
Ouro , Imidazóis , Nanopartículas Metálicas , Oligopeptídeos , Oxigenases , Ouro/química , Cobre/química , Nanopartículas Metálicas/química , Oxirredução , Minerais , Metano/química , Eletrodos
19.
Planta ; 259(4): 84, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38448635

RESUMO

MAIN CONCLUSION: A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.


Assuntos
Dianthus , Oxigenases , Humanos , Edição de Genes , RNA Guia de Sistemas CRISPR-Cas , Melhoramento Vegetal , Eletroporação , Ribonucleoproteínas
20.
Proc Natl Acad Sci U S A ; 121(11): e2308570121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38442170

RESUMO

Cytokinesis is the last step of cell division and is regulated by the small GTPase RhoA. RhoA activity is required for all steps of cytokinesis, including prior to abscission when daughter cells are ultimately physically separated. Like germ cells in all animals, the Caenorhabditis elegans embryonic germline founder cell initiates cytokinesis but does not complete abscission, leaving a stable intercellular bridge between the two daughter cells. Here, we identify and characterize C. elegans OSGN-1 as a cytokinetic regulator that promotes RhoA activity during late cytokinesis. Sequence analyses and biochemical reconstitutions reveal that OSGN-1 is a flavin-containing monooxygenase (MO). Genetic analyses indicate that the MO activity of OSGN-1 is required to maintain active RhoA at the end of cytokinesis in the germline founder cell and to stabilize the intercellular bridge. Deletion of OSGIN1 in human cells results in an increase in binucleation as a result of cytokinetic furrow regression, and this phenotype can be rescued by expressing a catalytically active form of C. elegans OSGN-1, indicating that OSGN-1 and OSGIN1 are functional orthologs. We propose that OSGN-1 and OSGIN1 are conserved MO enzymes required to maintain RhoA activity at the intercellular bridge during late cytokinesis and thus favor its stability, enabling proper abscission in human cells and bridge stabilization in C. elegans germ cells.


Assuntos
Citocinese , Dermatite , Oxigenases , Animais , Humanos , Citocinese/genética , Caenorhabditis elegans/genética , Divisão Celular
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