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1.
Inorg Chem ; 60(14): 10199-10214, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34213893

RESUMO

Two homologous 2-oxoglutarate-dependent (ODD) nonheme enzymes thebaine 6-O-demethylase (T6ODM) and codeine-3-O-demethylase (CODM), are involved in the morphine biosynthesis pathway from thebaine, catalyzing the O-demethylation reaction with precise regioselectivity at C6 and C3 positions of thebaine respectively. We investigated the origin of the regioselectivity of these enzymes by combined molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations and found that Thebaine binds at the two distinct sites of T6ODM and CODM, which determines the regioselectivity of the enzymes. A remarkable oxo rotation is observed in the decarboxylation process. Starting from the closed pentacoordinate configuration, the C-terminal lid adopts an open conformation in the octahedral Fe(IV) = O complex to facilitate the subsequent demethylation. Phe241 and Phe311 stabilize the substrate in the binding pocket, while Arg219 acts as a gatekeeper residue to stabilize the substrate. Our results unravel the regioselectivity in 2-OG dependent nonheme enzymes and may shed light for exploring the substrate scope of these enzymes and developing novel biotechnology for morphine biosynthesis.


Assuntos
Codeína/metabolismo , Simulação de Dinâmica Molecular , Oxirredutases O-Desmetilantes/metabolismo , Tebaína/química , Sítios de Ligação , Biocatálise , Metilação , Oxirredutases O-Desmetilantes/química , Conformação Proteica , Especificidade por Substrato
2.
Theor Appl Genet ; 134(8): 2379-2398, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34128089

RESUMO

KEY MESSAGE: Methyl esterase (MES), PvMES1, contributes to the defense response toward Fusarium wilt in common beans by regulating the salicylic acid (SA) mediated signaling pathway from phenylpropanoid synthesis and sugar metabolism as well as others. Common bean (Phaseolus vulgaris L.) is an important food legume. Fusarium wilt caused by Fusarium oxysporum f. sp. phaseoli is one of the most serious soil-borne diseases of common bean found throughout the world and affects the yield and quality of the crop. Few sources of Fusarium wilt resistance exist in legumes and most are of quantitative inheritance. In this study, we have identified a methyl esterase (MES), PvMES1, that contributes to plant defense response by regulating the salicylic acid (SA) mediated signaling pathway in response to Fusarium wilt in common beans. The result showed the role of PvMES1 in regulating SA levels in common bean and thus the SA signaling pathway and defense response mechanism in the plant. Overexpression of the PvMES1 gene enhanced Fusarium wilt resistance; while silencing of the gene caused susceptibility to the diseases. RNA-seq analysis with these transiently modified plants showed that genes related to SA level changes included the following gene ontologies: (a) phenylpropanoid synthesis; (b) sugar metabolism; and (c) interaction between host and pathogen as well as others. These key signal elements activated the defense response pathway in common bean to Fusarium wilt. Collectively, our findings indicate that PvMES1 plays a pivotal role in regulating SA biosynthesis and signaling, and increasing Fusarium wilt resistance in common bean, thus providing novel insight into the practical applications of both SA and MES genes and pathways they contribute to for developing elite crop varieties with enhanced broad-spectrum resistance to this critical disease.


Assuntos
Resistência à Doença/imunologia , Fusarium/fisiologia , Oxirredutases O-Desmetilantes/metabolismo , Phaseolus/imunologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Ácido Salicílico/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Oxirredutases O-Desmetilantes/genética , Phaseolus/genética , Phaseolus/crescimento & desenvolvimento , Phaseolus/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Transdução de Sinais
3.
Appl Environ Microbiol ; 86(22)2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-32917754

RESUMO

Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB The ferulate catabolism genes (ferBA), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX.IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.


Assuntos
Proteínas de Bactérias/genética , Ácido Gálico/análogos & derivados , Oxirredutases O-Desmetilantes/genética , Sphingomonadaceae/genética , Ácido Vanílico/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Gálico/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Sphingomonadaceae/metabolismo
4.
Int J Biol Macromol ; 163: 718-729, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32650010

RESUMO

Thebaine 6-O-demethylase (T6ODM) is an Fe(II)/2-oxoglutarate-dependent dioxygenase catalysing two oxidative O-demethylation reactions in morphine biosynthesis. Its crystal structure revealed a large active site pocket which is at least two times larger than necessary to accommodate a substrate (thebaine or oripavine) molecule. Since so far no crystal structures have been obtained for enzyme-substrate complex, which is necessary to explain the enzyme regiospecificity towards the C6-bound methoxy group, in this work we used computational methods and multi-parametric surface plasmon resonance measurements to elucidate the most likely structure of this complex and the reaction mechanism starting therefrom. Results of simulations and experiments unanimously indicate that the enzyme-substrate complex of T6ODM has a 1:2 stoichiometry. The key residues responsible for substrate binding are: Val-128, Glu-133, Met-150 and Agr-219 for the substrate in the distal position, and Asp-144, Leu-235 and Leu-353 for the proximal substrate molecule. QM/MM and DFT calculations show that the oxo ligand is bound trans to His-295 and the enzyme catalyzes hydroxylation of the C6-bound methoxy group according to the established rebound mechanism. The final stage of the demethylation reaction, which includes deformylation and enol-keton tautomerization steps, is most likely catalysed by water molecules and takes place in the solvent.


Assuntos
Oxirredutases O-Desmetilantes/química , Tebaína/química , Biocatálise , Teoria da Densidade Funcional , Hidroxilação , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Aquat Toxicol ; 225: 105540, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32569997

RESUMO

The zebrafish (Danio rerio) embryo has increasingly been used as an alternative model in human and environmental toxicology. Since the cytochrome P450 (CYP) system is of fundamental importance for the understanding and correct interpretation of the outcome of toxicological studies, constitutive and xenobiotic-induced 7-methoxycoumarin-O-demethylase (MCOD), i.e. 'mammalian CYP2-like', activities were monitored in vivo in zebrafish embryos via confocal laser scanning microscopy. In order to elucidate molecular mechanisms underlying the MCOD induction, dose-dependent effects of the prototypical CYP inducers ß-naphthoflavone (aryl hydrocarbon receptor (AhR) agonist), rifampicin (pregnane X receptor (PXR) agonist), carbamazepine and phenobarbital (constitutive androstane receptor (CAR) agonists) were analyzed in zebrafish embryos of varying age. Starting from 36 h of age, all embryonic stages of zebrafish could be shown to have constitutive MCOD activity, albeit with spatial variation and at distinct levels. Whereas carbamazepine, phenobarbital and rifampicin had no effect on in vivo MCOD activity in 96 h old zebrafish embryos, the model aryl hydrocarbon receptor agonist ß-naphthoflavone significantly induced MCOD activity in 96 h old zebrafish embryos at 46-734 nM, however, without a clear concentration-effect relationship. Induction of MCOD activity by ß-naphthoflavone gradually decreased with progression of embryonic development. By in vivo characterization of constitutive and xenobiotic-induced MCOD activity patterns in 36, 60, 84 and 108 h old zebrafish embryos, this decrease could primarily be attributed to an age-related decline in the induction of MCOD activity in the cardiovascular system. Results of this study provide novel insights into the mechanism and extent, by which specific CYP activities in early life-stages of zebrafish can be influenced by exposure to xenobiotics. The study thus lends further support to the view that zebrafish embryos- at least from an age of 36 h - have an elaborate and inducible biotransformation system.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Embrião não Mamífero/efeitos dos fármacos , Oxirredutases O-Desmetilantes/biossíntese , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Biotransformação , Indutores das Enzimas do Citocromo P-450/toxicidade , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Xenobióticos/toxicidade , Proteínas de Peixe-Zebra/metabolismo , beta-Naftoflavona/toxicidade
6.
Ecotoxicol Environ Saf ; 192: 110330, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32078841

RESUMO

Zebrafish (Danio rerio) early life-stages are increasingly gaining attention as an alternative model in both human and environmental toxicology. Whereas there is amble knowledge about the transcription of various cytochrome P450 isoforms, the level of information about functional implications is still limited. This study investigated the development of CYP2-dependent 7-methoxycoumarin-O-demethylase (MCOD) activity throughout the early zebrafish development from 5 to 118 h post-fertilization (hpf) via confocal laser scanning microscopy. Results demonstrate that zebrafish embryos exhibit constitutive MCOD activity from as early as 5.5 hpf. Characteristic spatiotemporal patterns were documented with MCOD activities localized in several tissues and organs, namely the cardiovascular system, the brain, the digestive system, and the urinary tract. The study thereby contributes to a better understanding of the development and functional role of CYP enzymes in zebrafish early life-stages.


Assuntos
Oxirredutases O-Desmetilantes/metabolismo , Peixe-Zebra/embriologia , Animais , Família 2 do Citocromo P450/metabolismo , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário , Fluorescência , Peixe-Zebra/metabolismo
7.
New Phytol ; 225(1): 234-249, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419316

RESUMO

Brown algae have convergently evolved plant-like body plans and reproductive cycles, which in plants are controlled by differential DNA methylation. This contribution provides the first single-base methylome profiles of haploid gametophytes and diploid sporophytes of a multicellular alga. Although only c. 1.4% of cytosines in Saccharina japonica were methylated mainly at CHH sites and characterized by 5-methylcytosine (5mC), there were significant differences between life-cycle stages. DNA methyltransferase 2 (DNMT2), known to efficiently catalyze tRNA methylation, is assumed to methylate the genome of S. japonica in the structural context of tRNAs as the genome does not encode any other DNA methyltransferases. Circular and long noncoding RNA genes were the most strongly methylated regulatory elements in S. japonica. Differential expression of genes was negatively correlated with DNA methylation with the highest methylation levels measured in both haploid gametophytes. Hypomethylated and highly expressed genes in diploid sporophytes included genes involved in morphogenesis and halogen metabolism. The data herein provide evidence that cytosine methylation, although occurring at a low level, is significantly contributing to the formation of different life-cycle stages, tissue differentiation and metabolism in brown algae.


Assuntos
Metilação de DNA/genética , Kelp/genética , Microalgas/genética , Plantas/genética , Cromossomos de Plantas/genética , Citosina/metabolismo , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Heterozigoto , Metiltransferases/genética , Metiltransferases/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Regiões Promotoras Genéticas/genética , Transcriptoma/genética
8.
Sci Rep ; 9(1): 18036, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792252

RESUMO

Vanillate and syringate are major intermediate metabolites generated during the microbial degradation of lignin. In Sphingobium sp. SYK-6, vanillate is O demethylated to protocatechuate by LigM; protocatechuate is then catabolized via the protocatechuate 4,5-cleavage pathway. Syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and then gallate is subjected to ring cleavage by DesB. Here, we investigated the transcriptional regulation of desA, ligM, and desB involved in vanillate and syringate catabolism. Quantitative reverse transcription-PCR analyses indicated that the transcription of these genes was induced 5.8-37-fold in the presence of vanillate and syringate. A MarR-type transcriptional regulator, SLG_12870 (desR), was identified as the gene whose product bound to the desB promoter region. Analysis of a desR mutant indicated that the transcription of desB, ligM, and desR is negatively regulated by DesR. Purified DesR bound to the upstream regions of desB, ligM, and desR, and the inverted repeat sequences similar to each other in these regions were suggested to be essential for DNA binding of DesR. Vanillate and syringate inhibited DNA binding of DesR, indicating that these compounds are effector molecules of DesR. The transcription of desA was found to be regulated by an as-yet unidentified regulator.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Lignina/metabolismo , Proteínas Repressoras/metabolismo , Sphingomonadaceae/fisiologia , Proteínas de Bactérias/genética , Redes e Vias Metabólicas/genética , Oxirredutases O-Desmetilantes/genética , Oxirredutases O-Desmetilantes/metabolismo , Regiões Promotoras Genéticas/genética , Transcrição Genética , Ácido Vanílico/metabolismo
9.
Proc Natl Acad Sci U S A ; 116(28): 13970-13976, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31235604

RESUMO

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Lignina/genética , Engenharia de Proteínas , Pirogalol/análogos & derivados , Sistema Enzimático do Citocromo P-450/química , Lignina/biossíntese , Lignina/metabolismo , Metilação , Oxirredução , Oxirredutases O-Desmetilantes/química , Oxirredutases O-Desmetilantes/genética , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Pirogalol/química , Pirogalol/metabolismo
10.
J Ind Microbiol Biotechnol ; 46(8): 1071-1080, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31134414

RESUMO

Microbial production of cis,cis-muconate (ccMA) from phenolic compounds obtained by chemical depolymerization of lignin is a promising approach to valorize lignin. Because microbial production requires a large amount of carbon and energy source, it is desirable to establish a ccMA-producing strain that utilizes lignin-derived phenols instead of general sources like glucose. We isolated Pseudomonas sp. strain NGC7 that grows well on various phenolic compounds derived from p-hydroxyphenyl, guaiacyl, and syringyl units of lignin. An NGC7 mutant of protocatechuate (PCA) 3,4-dioxygenase and ccMA cycloisomerase genes (NGC703) lost the ability to grow on vanillate and p-hydroxybenzoate but grew normally on syringate. Introduction of a plasmid carrying genes encoding PCA decarboxylase, flavin prenyltransferase, vanillate O-demethylase, and catechol 1,2-dioxygenase into NGC703 enabled production of 3.2 g/L ccMA from vanillate with a yield of 75% while growing on syringate. This strain also produced ccMA from birch lignin-derived phenols. All these results indicate the utility of NGC7 in glucose-free ccMA production.


Assuntos
Lignina/metabolismo , Pseudomonas/metabolismo , Ácido Sórbico/análogos & derivados , Catecóis/metabolismo , Glucose/metabolismo , Liases Intramoleculares/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Plasmídeos/genética , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Ácido Sórbico/metabolismo
11.
J Med Microbiol ; 68(6): 952-956, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31107204

RESUMO

Exploiting the immunosuppressive, analgesic and highly addictive properties of morphine could increase the success of a bacterial pathogen. Therefore, we performed sequence similarity searches for two morphine biosynthesis demethylases in bacteria. For thebaine 6-O-demethylase and codeine O-demethylase, we found strong alignments to three (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) of the six ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) that are commonly associated with drug resistance and nosocomial infections. Expression of the aligned sequence found in P. aeruginosa (NP_252880.1/PA4191) is upregulated in isolates obtained from cystic fibrosis patients. Our findings provide putative mechanistic targets for understanding the role of morphine in pathogenicity.


Assuntos
Acinetobacter baumannii/enzimologia , Infecção Hospitalar/microbiologia , Enterobacter/enzimologia , Klebsiella pneumoniae/enzimologia , Oxirredutases O-Desmetilantes/genética , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Acinetobacter baumannii/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Codeína/metabolismo , Enterobacter/genética , Humanos , Klebsiella pneumoniae/genética , Derivados da Morfina/metabolismo , Alcaloides Opiáceos/administração & dosagem , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Staphylococcus aureus/genética , Tebaína/metabolismo
12.
Xenobiotica ; 49(10): 1133-1142, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30392427

RESUMO

The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions. Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis-Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot. Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (∼50-fold) affinity but a higher (∼six-fold) capacity. The apparent CYP2D affinity and capacity of the crude mitochondria were in between those of the microsomes and purified mitochondria. Additionally, the CYP2D activity in the whole homogenate was much higher than that in the total membranes at higher DXM concentrations. A CYP2D immune-reactive band in the brain mitochondria appeared at a lower MW but had a much higher intensity than that in the microsomes. Mitochondrial brain CYP2D has a much higher capacity than its microsomal counterpart. Additionally, brain homogenate is more representative of the overall CYP2D activity than the widely-used total membrane fraction.


Assuntos
Encéfalo/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/metabolismo , Microssomos/enzimologia , Mitocôndrias/enzimologia , Oxirredutases O-Desmetilantes/metabolismo , Animais , Química Encefálica , Sistema Enzimático do Citocromo P-450/química , Família 2 do Citocromo P450/química , Cinética , Masculino , Oxirredutases O-Desmetilantes/química , Ratos , Ratos Sprague-Dawley
13.
Artigo em Inglês | MEDLINE | ID: mdl-30149296

RESUMO

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2 µL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 µL), microsomal protein amount (5 µg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ±â€¯SD, n = 4), which showed a maximum velocity of 2.24 ±â€¯0.42 pmol/min/mg and a MM constant of 282 ±â€¯62 µM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.


Assuntos
Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Dextrometorfano/metabolismo , Microssomos/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Animais , Encéfalo/citologia , Desmetilação , Dextrometorfano/análise , Dextrorfano/análise , Dextrorfano/metabolismo , Cinética , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos
14.
J Microbiol Methods ; 152: 126-134, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30076868

RESUMO

The ability of enzymatic Kraft Lignin (KL) demethylation was determined using catechol and ferric ion coordination (catechol-Fe3+ complexes) by reduction of Fe3+ to Fe2+ and formation of mono, bis- and/or tris-catechol-Fe3+ complexes has been investigated to identify enzyme that can strip-off O-methyl groups from lignin such as O-demethylase. To detect fungal demethylation and release of catechol-like structures, these were demonstrated using catechol, gallic acid and caffeic acid as standard model compounds to forms mono, bis- and/or tris-catechol-Fe3+ complexes. The catechol-Fe3+ complexes formation controlled by pH via the deprotonation of the catechol hydroxyls was investigated at pH 2.5, 8.0 and 10.0 and demonstrated that catechol formed mono, bis- and/or tris-catechol-Fe3+ complexes, and showed maximum absorbance at 547 nm. Lignin demethylation (O-demethylase) and formation of pyrocatecholic structures was detected using Aspergillus sp. and Galerina autumnalis culture filtrates as the enzyme source. The produced aromatic vicinal diol groups in lignin model compounds (LMCs) and KL were determined using different catecholic-binding reagents with the influence of H2O2, along with 4-antiaminopyrine reagent, was analyzed by the following: i) Fe3+-catechol complexation method, ii) HNO2 method, iii) FAS (Ferric Ammonium-Sulfate) method, iv) Ti(III)-NTA (Titanium (III)- Nitrilotriacetate) method for hydrolytic zone formation. Among the tested methods showing lytic zone formation was Fe3+-catechol complexation. The LMCs and KL treated using Aspergillus sp. culture filtrate showed maximum Fe3+-catechol complexes with 3-methoxy catechol (91 µmol/mL), o-vanillin (44 µmol/mL) and KL (100 µmol/mL). In addition, Galerina autumnalis culture filtrate showed demethylation of vanillin (48 µmol/mL), 3-methoxy catechol (82 µmol/mL), o-vanillin, (33 µmol/mL), 3 4-dimethoxybenzyl alcohol (49 µmol/mL) and KL (41 µmol/mL). The results suggest that lignin demethylation (O-demethylases) activity that strip-off methyl groups in LMCs and KL and produced vicinal diols that covalently bind with Fe3+ to form Fe3+-catechol complexes. The new Fe3+-catechol complexation method has the ability to characterize pyrocatechol and galloyl structures in chemically or biologically modified lignins and to detect O-demethylase activity.


Assuntos
Catecóis/metabolismo , Desmetilação , Ensaios Enzimáticos/métodos , Ferro/metabolismo , Lignina/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Aspergillus/enzimologia , Aspergillus/metabolismo , Basidiomycota/enzimologia , Basidiomycota/metabolismo , Benzaldeídos/metabolismo , Ácidos Cafeicos , Catálise , Compostos Férricos , Fungos/enzimologia , Fungos/metabolismo , Ácido Gálico , Concentração de Íons de Hidrogênio , Madeira/metabolismo , Madeira/microbiologia
15.
Mol Plant ; 11(10): 1264-1277, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30055263

RESUMO

Nicotinamide adenine dinucleotide (NAD) biosynthesis, including synthesis from aspartate via the de novo pathway and from nicotinate (NA) via the Preiss-Handler pathway, is conserved in land plants. Diverse species of NA conjugates, which are mainly involved in NA detoxification, were also found in all tested land plants. Among these conjugates, MeNA (NA methyl ester) has been widely detected in angiosperm plants, although its physiological function and the underlying mechanism for its production in planta remain largely unknown. Here, we show that MeNA is an NAD precursor undergoing more efficient long-distance transport between organs than NA and nicotinamide in Arabidopsis. We found that Arabidopsis has one methyltransferase (designated AtNaMT1) capable of catalyzing carboxyl methylation of NA to yield MeNA and one methyl esterase (MES2) predominantly hydrolyzing MeNA back to NA. We further uncovered that the transfer of [14C]MeNA from the root to leaf was significantly increased in both MES2 knockdown and NaMT1-overexpressing lines, suggesting that both NaMT1 and MES2 fine-tune the long-distance transport of MeNA, which is ultimately utilized for NAD production. Abiotic stress (salt, abscisic acid, and mannitol) treatments, which are known to exacerbate NAD degradation, induce the expression of NaMT1 but suppress MES2 expression, suggesting that MeNA may play a role in stress adaption. Collectively, our study indicates that reversible methylation of NA controls the biosynthesis of MeNA in Arabidopsis, which presumably functions as a detoxification form of free NA for efficient long-distance transport and eventually NAD production especially under abiotic stress, providing new insights into the relationship between NAD biosynthesis and NA conjugation in plants.


Assuntos
Arabidopsis/metabolismo , NAD/metabolismo , Niacina/metabolismo , Ácidos Nicotínicos/metabolismo , Arabidopsis/enzimologia , Transporte Biológico , Metilação , Oxirredutases O-Desmetilantes/metabolismo , Estresse Fisiológico
16.
Chem Asian J ; 13(19): 2854-2867, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-29917331

RESUMO

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self-assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic "thermal tag" with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes: FTO (7; IC50 =2.6 µm) and ALKBH3 (8; IC50 =3.7 µm). To date, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.


Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Dioxigenase FTO Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Técnicas de Química Combinatória/métodos , Inibidores Enzimáticos/química , Oxirredutases O-Desmetilantes/antagonistas & inibidores , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/química , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/química , Homólogo AlkB 5 da RNA Desmetilase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Catálise , Fluorometria/métodos , Humanos , Hidrazonas/química , Cinética , Ligantes , Estrutura Molecular , Oxirredutases O-Desmetilantes/química , Oxirredutases O-Desmetilantes/genética , Peptídeos/química , Peptídeos/genética , Desnaturação Proteica , Engenharia de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Temperatura de Transição
17.
Nat Commun ; 9(1): 2487, 2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29950589

RESUMO

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion.


Assuntos
Actinobacteria/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Lignina/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Oxirredução , Oxirredutases O-Desmetilantes/química , Multimerização Proteica , Especificidade por Substrato
18.
J Struct Biol ; 202(3): 229-235, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29408320

RESUMO

Thebaine 6-O-demethylase (T6ODM) from Papaver somniferum (opium poppy), which belongs to the non-heme 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODD) family, is a key enzyme in the morphine biosynthesis pathway. Initially, T6ODM was characterized as an enzyme catalyzing O-demethylation of thebaine to neopinone and oripavine to morphinone. However, the substrate range of T6ODM was recently expanded to a number of various benzylisoquinoline alkaloids. Here, we present crystal structures of T6ODM in complexes with 2-oxoglutarate (T6ODM:2OG, PDB: 5O9W) and succinate (T6ODM:SIN, PDB: 5O7Y). Both metal and 2OG binding sites display similarity to other proteins from the ODD family, but T6ODM is characterized by an exceptionally large substrate binding cavity, whose volume can partially explain the promiscuity of this enzyme. Moreover, the size of the cavity allows for binding of multiple molecules at once, posing a question about the substrate-driven specificity of the enzyme.


Assuntos
Oxirredutases O-Desmetilantes/ultraestrutura , Papaver/enzimologia , Tebaína/química , Cristalografia por Raios X , Ácidos Cetoglutáricos/química , Metilação , Morfina/biossíntese , Morfina/química , Oxirredutases O-Desmetilantes/química , Papaver/química , Ácido Succínico/química
19.
Plant Cell Environ ; 40(11): 2806-2819, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28810288

RESUMO

The transition from active growth to dormancy is critical for the survival of perennial plants. We identified a DEMETER-like (CsDML) cDNA from a winter-enriched cDNA subtractive library in chestnut (Castanea sativa Mill.), an economically and ecologically important species. Next, we characterized this DNA demethylase and its putative ortholog in the more experimentally tractable hybrid poplar (Populus tremula × alba), under the signals that trigger bud dormancy in trees. We performed phylogenetic and protein sequence analysis, gene expression profiling, and 5-methyl-cytosine methylation immunodetection studies to evaluate the role of CsDML and its homolog in poplar, PtaDML6. Transgenic hybrid poplars overexpressing CsDML were produced and analysed. Short days and cold temperatures induced CsDML and PtaDML6. Overexpression of CsDML accelerated short-day-induced bud formation, specifically from Stages 1 to 0. Buds acquired a red-brown coloration earlier than wild-type plants, alongside with the up-regulation of flavonoid biosynthesis enzymes and accumulation of flavonoids in the shoot apical meristem and bud scales. Our data show that the CsDML gene induces bud formation needed for the survival of the apical meristem under the harsh conditions of winter.


Assuntos
Meristema/enzimologia , Meristema/crescimento & desenvolvimento , Oxirredutases O-Desmetilantes/metabolismo , Proteínas de Plantas/metabolismo , Populus/enzimologia , Populus/crescimento & desenvolvimento , Sequência de Aminoácidos , Arabidopsis/genética , Domínio Catalítico , Temperatura Baixa , DNA Glicosilases/química , DNA Glicosilases/metabolismo , Metilação de DNA/genética , Flavonoides/metabolismo , Fluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hippocastanaceae/enzimologia , Hippocastanaceae/genética , Hippocastanaceae/crescimento & desenvolvimento , Meristema/genética , Fotoperíodo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Populus/genética , Estações do Ano
20.
Proc Natl Acad Sci U S A ; 114(16): E3205-E3214, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28373573

RESUMO

Some strains of soil and marine bacteria have evolved intricate metabolic pathways for using environmentally derived aromatics as a carbon source. Many of these metabolic pathways go through intermediates such as vanillate, 3-O-methylgallate, and syringate. Demethylation of these compounds is essential for downstream aryl modification, ring opening, and subsequent assimilation of these compounds into the tricarboxylic acid (TCA) cycle, and, correspondingly, there are a variety of associated aryl demethylase systems that vary in complexity. Intriguingly, only a basic understanding of the least complex system, the tetrahydrofolate-dependent aryl demethylase LigM from Sphingomonas paucimobilis, a bacterial strain that metabolizes lignin-derived aromatics, was previously available. LigM-catalyzed demethylation enables further modification and ring opening of the single-ring aromatics vanillate and 3-O-methylgallate, which are common byproducts of biofuel production. Here, we characterize aryl O-demethylation by LigM and report its 1.81-Å crystal structure, revealing a unique demethylase fold and a canonical folate-binding domain. Structural homology and geometry optimization calculations enabled the identification of LigM's tetrahydrofolate-binding site and protein-folate interactions. Computationally guided mutagenesis and kinetic analyses allowed the identification of the enzyme's aryl-binding site location and determination of its unique, catalytic tyrosine-dependent reaction mechanism. This work defines LigM as a distinct demethylase, both structurally and functionally, and provides insight into demethylation and its reaction requirements. These results afford the mechanistic details required for efficient utilization of LigM as a tool for aryl O-demethylation and as a component of synthetic biology efforts to valorize previously underused aromatic compounds.


Assuntos
Oxirredutases O-Desmetilantes/química , Oxirredutases O-Desmetilantes/metabolismo , Sphingomonas/enzimologia , Tirosina/metabolismo , Catálise , Cristalografia por Raios X , Cinética , Redes e Vias Metabólicas , Conformação Proteica
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