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1.
Tidsskr Nor Laegeforen ; 1412021 09 28.
Artigo em Inglês, Norueguês | MEDLINE | ID: mdl-34597008

RESUMO

BACKGROUND: Trimethylaminuria is a rare disorder characterised by foul odour from bodily fluids and breath. The condition is caused by a homozygous mutation in the FMO3 (flavin monooxygenase 3) gene coding for the enzyme that converts TMA (trimethylamine) to trimethylamine N-oxide. The result is elevated levels of secreted trimethylamine, which has a strong odour. The condition is likely to affect mental, emotional and social health. The diagnosis is reached by testing of free TMA (trimethylamine) and percentage N-oxidation in urine samples or by genetic testing. CASE PRESENTATION: A man in his fifties had from childhood occasionally been told that his breath resembled rotten fish. He had searched for a diagnosis on the internet and was referred to testing for trimethylaminuria, and the diagnosis was confirmed. INTERPRETATION: Urine test samples with high levels of free TMA and subnormal percentage of trimethylamine N-oxide revealed the diagnosis of trimethylaminuria. There is no causal treatment. Patients are advised to avoid choline-rich foods and take hygienic measures.


Assuntos
Erros Inatos do Metabolismo , Oxigenases , Animais , Criança , Peixes , Humanos , Masculino , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Metilaminas/urina , Mutação , Oxigenases/genética
2.
Microbes Environ ; 36(3)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34511574

RESUMO

Bradyrhizobium sp. RD5-C2, isolated from soil that is not contaminated with 2,4-dichlorophenoxyacetic acid (2,4-D), degrades the herbicides 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). It possesses tfdAα and cadA (designated as cadA1), which encode 2,4-D dioxygenase and the oxygenase large subunit, respectively. In the present study, the genome of Bradyrhizobium sp. RD5-C2 was sequenced and a second cadA gene (designated as cadA2) was identified. The two cadA genes belonged to distinct clusters comprising the cadR1A1B1K1C1 and cadR2A2B2C2K2S genes. The proteins encoded by the cad1 cluster exhibited high amino acid sequence similarities to those of other 2,4-D degraders, while Cad2 proteins were more similar to those of non-2,4-D degraders. Both cad clusters were capable of degrading 2,4-D and 2,4,5-T when expressed in non-2,4-D-degrading Bradyrhizobium elkanii USDA94. To examine the contribution of each degradation gene cluster to the degradation activity of Bradyrhizobium sp. RD5-C2, cadA1, cadA2, and tfdAα deletion mutants were constructed. The cadA1 deletion resulted in a more significant decrease in the ability to degrade chlorophenoxy compounds than the cadA2 and tfdAα deletions, indicating that degradation activity was primarily governed by the cad1 cluster. The results of a quantitative reverse transcription-PCR analysis suggested that exposure to 2,4-D and 2,4,5-T markedly up-regulated cadA1 expression. Collectively, these results indicate that the cad1 cluster plays an important role in the degradation of Bradyrhizobium sp. RD5-C2 due to its high expression.


Assuntos
Ácido 2,4-Diclorofenoxiacético/metabolismo , Proteínas de Bactérias/genética , Bradyrhizobium/metabolismo , Herbicidas/metabolismo , Família Multigênica , Ácido 2,4-Diclorofenoxiacético/química , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Bradyrhizobium/classificação , Bradyrhizobium/enzimologia , Bradyrhizobium/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Herbicidas/química , Oxigenases/genética , Oxigenases/metabolismo , Filogenia , Microbiologia do Solo
3.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208368

RESUMO

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.


Assuntos
Alcaloides Indólicos/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases/metabolismo , Quinolinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Vimblastina/análogos & derivados , Vias Biossintéticas , Vimblastina/biossíntese , Vimblastina/química
4.
Nat Commun ; 12(1): 4158, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230497

RESUMO

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible ß-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.


Assuntos
Cicloexenos/síntese química , Cicloexenos/metabolismo , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/metabolismo , Vias Biossintéticas/genética , Catálise , Técnicas de Química Sintética , Compostos de Epóxi , Fermentação , Proteínas Fúngicas/genética , Modelos Moleculares , Estrutura Molecular , Oxigenases , Penicillium/genética , Penicillium/metabolismo
5.
Nat Commun ; 12(1): 4417, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285212

RESUMO

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.


Assuntos
Aspergillus/enzimologia , Biocatálise , Proteínas Fúngicas/metabolismo , Oxigenases/metabolismo , Peróxidos/metabolismo , Aspergillus/genética , Domínio Catalítico , Cristalografia por Raios X , Compostos Ferrosos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Ácidos Cetoglutáricos/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/metabolismo , Oxigenases/genética , Oxigenases/isolamento & purificação , Oxigenases/ultraestrutura , Conformação Proteica em Folha beta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Terpenos/metabolismo
6.
Biosens Bioelectron ; 192: 113488, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34265522

RESUMO

Screening inhibitors of flavin monooxygenase 3 (FMO3) is very important for treating trimethylamine N-oxide (TMAO) derived thrombotic diseases. Herein, focusing on Xuefu Zhuyu decoction (XFZYD) as a Chinese traditional medicine with antithrombotic efficacy, a facile and label-free fluorescence strategy was developed for evaluating the influence of the bioactive ingredients in XFZYD on FMO3 activity through indicator displacement assay. To this end, the optimized supramolecular host-guest (p-sulfonatocalix[4]arene-oxazine 1) reporter pair and FMO3 catalytic system were exploited to determine the influence of the bioactive compounds in XFZYD on the conversion from TMA to TMAO. From the nine compounds tested, naringin, paeoniflorin, ß-ecdysterone, 18ß-glycyrrhizic acid, amygdalin, albiflorin, and saikosaponin A downregulated FMO3 activity and reduced TMAO biosynthesis. Moreover, molecular docking was successfully applied to simulate the optimal conformation of a receptor-ligand complex between FMO3 and all tested compounds except for ß-ecdysterone. Therefore, this approach provides a novel and promising strategy for screening FMO3 inhibitors from Chinese traditional medicine by supramolecular sensing.


Assuntos
Técnicas Biossensoriais , Trombose , Humanos , Metilaminas , Simulação de Acoplamento Molecular , Oxigenases
7.
Biochemistry ; 60(32): 2492-2507, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34324302

RESUMO

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make antibiotics and other bioactive materials or degrade plant material, but we have a limited understanding of the breadth and depth of substrate space for these potent catalysts. Here we report steady-state and pre-steady-state kinetic analysis of dopamine derivatives substituted at the 6-position as substrates of L-DOPA dioxygenase, and an analysis of that activity as a function of the electron-withdrawing nature of the substituent. Steady-state and pre-steady-state kinetic data demonstrate the dopamines are impaired in binding and catalysis with respect to the cosubstrate molecular oxygen, which likely afforded spectroscopic observation of an early reaction intermediate, the semiquinone of dopamine. The reaction pathway of dopamine in the pre-steady state is consistent with a nonproductive mode of binding of oxygen at the active site. Despite these limitations, L-DOPA dioxygenase is capable of binding all of the dopamine derivatives and catalyzing multiple turnovers of ring cleavage for dopamine, 6-bromodopamine, 6-carboxydopamine, and 6-cyanodopamine. 6-Nitrodopamine was a single-turnover substrate. The variety of substrates accepted by the enzyme is consistent with an interplay of factors, including the capacity of the active site to bind large, negatively charged groups at the 6-position and the overall oxidizability of each catecholamine, and is indicative of the utility of extradiol cleavage in semisynthetic and bioremediation applications.


Assuntos
Dioxigenases/metabolismo , Dopamina/análogos & derivados , Levodopa/metabolismo , Catálise , Domínio Catalítico , Catecóis/química , Catecóis/metabolismo , Ciclização , Dioxigenases/química , Dopamina/síntese química , Dopamina/metabolismo , Cinética , Levodopa/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxigenases/química , Especificidade por Substrato
8.
J Appl Genet ; 62(4): 527-543, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34109531

RESUMO

Cotton that serves natural fiber for the textile industry is an important industrial crop. However, abiotic stress imposed a significant negative impact on yield and quality of cotton fiber. Carotenoid cleavage oxygenases (CCOs) that specifically catalyze the cleavage of carotenoid are essential for plant growth and development and abiotic stress response. While information of cotton CCOs and their potential functions in abiotic stress is still far from satisfactory, which imposes restrictions on application in genetic breeding for stress resistance. In this study, 15, 15, and 30 CCOs were identified from Gossypium arboreum, Gossypium raimondii, and Gossypium hirsutum, respectively. Phylogenetic relationship indicated that CCO genes could be classified into two groups (NCEDs and CCDs). Cis-elements prediction showed that there were 18 types of stress-related cis-elements in promoter regions. Analysis with transcriptome data revealed tissue-specific expression pattern of cotton CCOs. qRT-PCR analysis revealed only that GhNCED3a_A/D and GhNCED3c_A/D had strong response to drought, salt, and cold stress, while GhCCD1_A/D and GhCCD4_A showed relatively slight expression changes. Virus-induced gene silencing of GaNCED3a, the ortholog gene of GhNCED3a_A/D, suggested that silenced plants exhibited decreased resistance not only to drought but also to salt, with significantly reduced proline content, and high malondialdehyde content and water loss rate. In addition, stress response genes RD29A, DREB1A, and SOS1 significantly downregulated under drought and salt stress in silenced plants compared to control plants, indicating that GaNCED3a played an important role in drought and salt response. The results provided valuable insights into function analysis of cotton CCOs in abiotic stress response, and suggested potential benefit genes for stress-resistant breeding.


Assuntos
Secas , Gossypium , Regulação da Expressão Gênica de Plantas , Gossypium/genética , Gossypium/metabolismo , Oxigenases , Filogenia , Melhoramento Vegetal , Proteínas de Plantas/genética , Estresse Salino/genética , Estresse Fisiológico/genética
9.
Sci Rep ; 11(1): 12045, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103559

RESUMO

Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy [Formula: see text] of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.


Assuntos
Enoil-(Proteína de Transporte de Acila) Redutase (NADPH, B-Específica)/metabolismo , Escherichia coli/enzimologia , Hidrocarbonetos/química , Alcanos/metabolismo , Proteínas de Bactérias/metabolismo , Biocombustíveis , Biofísica , Biotecnologia , Cromatografia , Cromatografia Líquida , Dicroísmo Circular , Luz , Espectrometria de Massas , Simulação de Dinâmica Molecular , Oxigenases/química , Desnaturação Proteica , Dobramento de Proteína , Espalhamento de Radiação , Espectrometria de Fluorescência , Eletricidade Estática , Synechococcus/metabolismo , Temperatura , Ureia/química
10.
Biochemistry ; 60(25): 1995-2010, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34100595

RESUMO

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of oxygen from O2 to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using 19F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 µM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane.


Assuntos
Proteínas de Bactérias/metabolismo , Oxigenases/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Bactérias/química , Flúor/química , Cinética , Methylosinus trichosporium/enzimologia , Ressonância Magnética Nuclear Biomolecular , Oxigenases/química , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Triptofano/análogos & derivados , Triptofano/química
11.
J Plant Res ; 134(5): 1121-1138, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34037878

RESUMO

Reaumuria trigyna, a Tamaricaceae archaic recretohalophyte, is an important feral forage plant in the desert steppe of northwestern China. We identified two significantly differentially expressed leucoanthocyanidin dioxygenase genes (RtLDOX/RtLDOX2) and investigated the function and characteristics of RtLDOX2. RtLDOX2 from R. trigyna was rapidly upregulated by salt, drought, and abscisic acid, consistent with the stress-related cis-regulatory elements in the promoter region. Recombinant RtLDOX2 converted dihydrokaempferol to kaempferol in vitro, and was thus interchangeable with flavonol synthase, a dioxygenase in the flavonoid pathway. Transgenic plants overexpressing RtLDOX2 accumulated more anthocyanin and flavonols under abiotic stresses, speculating that RtLDOX2 may act as a multifunctional dioxygenase in the synthesis of anthocyanins and flavonols. Overexpression of RtLDOX2 enhanced the primary root length, biomass accumulation, and chlorophyll content of salt-, drought-, and ultraviolet-B-stressed transgenic Arabidopsis. Antioxidant enzyme activity; proline content; and expression of antioxidant enzyme, proline biosynthesis, and ion-transporter genes were increased in transgenic plants. Therefore, RtLDOX2 confers tolerance to abiotic stress on transgenic Arabidopsis by promoting the accumulation of anthocyanins and flavonols. This in turn increases reactive oxygen species scavenging and activates other stress responses, such as osmotic adjustment and ion transport, and so improves tolerance to abiotic stresses.


Assuntos
Tamaricaceae , Antocianinas , Secas , Flavonoides , Regulação da Expressão Gênica de Plantas , Oxigenases , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tolerância ao Sal , Estresse Fisiológico , Tamaricaceae/genética , Tamaricaceae/metabolismo
12.
Brain ; 144(5): 1422-1434, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33970200

RESUMO

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.


Assuntos
Oxigenases/genética , Paraplegia Espástica Hereditária/genética , Animais , Feminino , Humanos , Masculino , Camundongos , Mutação , Linhagem , Ratos , Peixe-Zebra
13.
Angew Chem Int Ed Engl ; 60(29): 15827-15831, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33973699

RESUMO

Aziridine is a characteristically reactive molecule with increased bioactivity due to its strained ring structure. Here, we investigated the biosynthesis of 2-aminoisobutyric acid (AIB) in Penicillium, and successfully reconstituted the three-step biosynthesis from L-Val to AIB in vitro. This previously unknown aziridine formation pathway proceeded with the non-heme iron and α-ketoglutarate-dependent (FeII /αKG) oxygenase TqaL, followed by aziridine ring opening by the haloalkanoic acid dehalogenase (HAD)-type hydrolase TqaF, and subsequent oxidative decarboxylation by the NovR/CloR-like non-heme iron oxygenase TqaM. Furthermore, the X-ray crystal structure of the C-N bond forming FeII /αKG oxygenase TqaL was solved at 2.0 Šresolution. This work presents the first molecular basis for aziridine biogenesis, thereby expanding the catalytic repertoire of the FeII /αKG oxygenases. We also report the unique aziridine ring opening by a HAD-type hydrolase and the remarkable oxidative decarboxylation by a non-heme iron oxygenase to produce AIB.


Assuntos
Ácidos Aminoisobutíricos/metabolismo , Aziridinas/metabolismo , Fungos/metabolismo , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenases/metabolismo , Cinética , Oxirredução
15.
Microb Cell Fact ; 20(1): 107, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34044834

RESUMO

Uprising fossil fuel depletion and deterioration of ecological reserves supply have led to the search for alternative renewable and sustainable energy sources and chemicals. Although first generation biorefinery is quite successful commercially in generating bulk of biofuels globally, the food versus fuel debate has necessitated the use of non-edible feedstocks, majorly waste biomass, for second generation production of biofuels and chemicals. A diverse class of microbes and enzymes are being exploited for biofuels production for a series of treatment process, however, the conversion efficiency of wide range of lignocellulosic biomass (LCB) and consolidated way of processing remains challenging. There were lot of research efforts in the past decade to scour for potential microbial candidate. In this context, evolution has developed the gut microbiota of several insects and ruminants that are potential LCB degraders host eco-system to overcome its host nutritional constraints, where LCB processed by microbiomes pretends to be a promising candidate. Synergistic microbial symbionts could make a significant contribution towards recycling the renewable carbon from distinctly abundant recalcitrant LCB. Several studies have assessed the bioprospection of innumerable gut symbionts and their lignocellulolytic enzymes for LCB degradation. Though, some reviews exist on molecular characterization of gut microbes, but none of them has enlightened the microbial community design coupled with various LCB valorization which intensifies the microbial diversity in biofuels application. This review provides a deep insight into the significant breakthroughs attained in enrichment strategy of gut microbial community and its molecular characterization techniques which aids in understanding the holistic microbial community dynamics. Special emphasis is placed on gut microbial role in LCB depolymerization strategies to lignocellulolytic enzymes production and its functional metagenomic data mining eventually generating the sugar platform for biofuels and renewable chemicals production.


Assuntos
Biocombustíveis , Carbono/metabolismo , Microbioma Gastrointestinal , Lignina/metabolismo , Simbiose , Animais , Biomassa , Celulase , Fermentação , Microbiologia Industrial , Insetos/microbiologia , Oxigenases , Ruminantes/microbiologia
16.
PLoS Genet ; 17(5): e1009530, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33983934

RESUMO

Hadal environments (depths below 6,000 m) are characterized by extremely high hydrostatic pressures, low temperatures, a scarce food supply, and little light. The evolutionary adaptations that allow vertebrates to survive in this extreme environment are poorly understood. Here, we constructed a high-quality reference genome for Yap hadal snailfish (YHS), which was captured at a depth of ~7,000 m in the Yap Trench. The final YHS genome assembly was 731.75 Mb, with a contig N50 of 0.75 Mb and a scaffold N50 of 1.26 Mb. We predicted 24,329 protein-coding genes in the YHS genome, and 24,265 of these genes were successfully functionally annotated. Phylogenetic analyses suggested that YHS diverged from a Mariana Trench snailfish approximately 0.92 million years ago. Many genes associated with DNA repair show evidence of positive selection and have expanded copy numbers in the YHS genome, possibly helping to maintain the integrity of DNA under increased hydrostatic pressure. The levels of trimethylamine N-oxide (TMAO), a potent protein stabilizer, are much higher in the muscles of YHS than in those of shallow-water fish. This difference is perhaps due to the five copies of the TMAO-generating enzyme flavin-containing monooxygenase-3 gene (fmo3) in the YHS genome and the abundance of trimethylamine (TMA)-generating bacteria in the YHS gut. Thus, the high TMAO content might help YHS adapt to high hydrostatic pressure by improving protein stability. Additionally, the evolutionary features of the YHS genes encoding sensory-related proteins are consistent with the scarce food supply and darkness in the hadal environments. These results clarify the molecular mechanisms underlying the adaptation of hadal organisms to the deep-sea environment and provide valuable genomic resources for in-depth investigations of hadal biology.


Assuntos
Aclimatação/genética , Ambientes Extremos , Peixes/genética , Genoma/genética , Oceanos e Mares , Sequenciamento Completo do Genoma , Animais , Reparo do DNA/genética , Escuridão , Evolução Molecular , Peixes/classificação , Pressão Hidrostática , Metilaminas/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Filogenia , Estabilidade Proteica
17.
Elife ; 102021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33978570

RESUMO

The model organism Caenorhabditis elegans mounts transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, the regulation of which is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature, which was almost completely abrogated by deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Moreover, fmo-2/FMO5 induction required the nuclear hormone receptor, NHR-49/PPAR-α, which controlled host defense cell non-autonomously. These findings reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal FMOs as important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPAR-α during S. aureus infection, with implications for host defense and inflammation in higher organisms.


Assuntos
Caenorhabditis elegans/imunologia , Imunidade Inata , Oxigenases/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/metabolismo , Privação de Alimentos , Oxigenases/genética , PPAR alfa/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia
18.
Acc Chem Res ; 54(9): 2185-2195, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33886257

RESUMO

Rigorous substrate selectivity is a hallmark of enzyme catalysis. This selectivity is generally ascribed to a thermodynamically favorable process of substrate binding to the enzyme active site based upon complementary physiochemical characteristics, which allows both acquisition and orientation. However, this chemical selectivity is more difficult to rationalize for diminutive molecules that possess too narrow a range of physical characteristics to allow either precise positioning or discrimination between a substrate and an inhibitor. Foremost among these small molecules are dissolved gases such as H2, N2, O2, CO, CO2, NO, N2O, NH3, and CH4 so often encountered in metalloenzyme catalysis. Nevertheless, metalloenzymes have evolved to metabolize these small-molecule substrates with high selectivity and efficiency.The soluble methane monooxygenase enzyme (sMMO) acts upon two of these small molecules, O2 and CH4, to generate methanol as part of the C1 metabolic pathway of methanotrophic organisms. sMMO is capable of oxidizing many alternative hydrocarbon substrates. Remarkably, however, it will preferentially oxidize methane, the substrate with the fewest discriminating physical characteristics and the strongest C-H bond. Early studies led us to broadly attribute this specificity to the formation of a "molecular sieve" in which a methane- and oxygen-sized tunnel provides a size-selective route from bulk solvent to the completely buried sMMO active site. Indeed, recent cryogenic and serial femtosecond ambient temperature crystallographic studies have revealed such a route in sMMO. A detailed study of the sMMO tunnel considered here in the context of small-molecule tunnels identified in other metalloenzymes reveals three discrete characteristics that contribute to substrate selectivity and positioning beyond that which can be provided by the active site itself. Moreover, the dynamic nature of many tunnels allows an exquisite coordination of substrate binding and reaction phases of the catalytic cycle. Here we differentiate between the highly selective molecular tunnel, which allows only the one-dimensional transit of small molecules, and the larger, less-selective channels found in typical enzymes. Methods are described to identify and characterize tunnels as well as to differentiate them from channels. In metalloenzymes which metabolize dissolved gases, we posit that the contribution of tunnels is so great that they should be considered to be extensions of the active site itself. A full understanding of catalysis by these enzymes requires an appreciation of the roles played by tunnels. Such an understanding will also facilitate the use of the enzymes or their synthetic mimics in industrial or pharmaceutical applications.


Assuntos
Metaloproteínas/metabolismo , Oxigenases/química , Bibliotecas de Moléculas Pequenas/metabolismo , Biocatálise , Domínio Catalítico , Metaloproteínas/química , Modelos Moleculares , Oxigenases/metabolismo , Bibliotecas de Moléculas Pequenas/química
19.
J Ind Microbiol Biotechnol ; 48(3-4)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-33928356

RESUMO

Terpenoids are one of the largest classes of natural products whose members possess a wide variety of biological activities. With several exceptions, scalable production of complex terpenoids with either purely biological or chemical methods still remains a major challenge. However, recent efforts to combine the two approaches in chemoenzymatic synthesis hold tremendous promise to address this challenge. Central to this paradigm is the development of useful biocatalytic methods, such as regioselective C-H oxidation, for terpene modifications. This review highlights recent applications of biocatalytic hydroxylation for site-selective modification of terpenoids.


Assuntos
Oxigenases/metabolismo , Terpenos/metabolismo , Biocatálise , Hidroxilação , Oxirredução
20.
Int J Legal Med ; 135(4): 1471-1476, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33928430

RESUMO

N-Methyl-1-(naphthalen-2-yl)propan-2-amine (methamnetamine, PAL-1046) is an amphetamine-based new psychoactive substance (NPS). Methamnetamine has been reported to cause excessive release of serotonin, and it is classified as an empathogen or entactogen. It is not regulated as a controlled substance in most countries, and there are no studies on its metabolism. In this study, in vitro phase I metabolism of methamnetamine in human liver microsomes (HLM) and flavin-containing monooxygenase (FMO) was investigated by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). Eight metabolites of methamnetamine were identified and were structurally characterized achieved by a combination of accurate mass analysis and tandem mass spectrometry. The identified metabolic processes include N-demethylation, N-hydroxylation, aromatic hydroxylation, and a combination of these processes. N-Hydroxylated metabolites were confirmed based on expressed FMOs. The major metabolite was formed from methamnetamine via hydroxylation of the naphthalene ring after the in vitro phase I process. These results could help detect methamnetamine ingestion by NPS abusers.


Assuntos
Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Detecção do Abuso de Substâncias/métodos , Cromatografia Líquida , Desmetilação , Humanos , Hidroxilação , Técnicas In Vitro , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
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