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1.
J Agric Food Chem ; 67(31): 8573-8580, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31293156

RESUMO

Glycosylation endows both natural and synthetic small molecules with modulated physicochemical and biological properties. Plant and bacterial glycosyltransferases capable of decorating various privileged scaffolds have been extensively studied, but those from kingdom Fungi still remain underexploited. Here, we use a combination of genome mining and heterologous expression techniques to identify four novel glycosyltransferase-methyltransferase (GT-MT) functional modules from Hypocreales fungi. These GT-MT modules display decent substrate promiscuity and regiospecificity, methylglucosylating a panel of natural products such as flavonoids, stilbenoids, anthraquinones, and benzenediol lactones. Native GT-MT modules can be split up and regrouped into hybrid modules with similar or even improved efficacy as compared with native pairs. Methylglucosylation of kaempferol considerably improves its insecticidal activity against the larvae of oriental armyworm Mythimna separata (Walker). Our work provides a set of efficient biocatalysts for the combinatorial biosynthesis of small molecule glycosides that may have significant importance to the pharmaceutical, agricultural, and food industries.


Assuntos
Proteínas Fúngicas/química , Glicosiltransferases/química , Hypocreales/enzimologia , Metiltransferases/química , Fenóis/química , Animais , Biocatálise , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hypocreales/genética , Inseticidas/química , Inseticidas/farmacologia , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mariposas/efeitos dos fármacos , Fenóis/farmacologia , Especificidade por Substrato
2.
Food Chem Toxicol ; 131: 110529, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150784

RESUMO

The health promoting effects of extra virgin olive oil (EVOO) relate to its unique repertoire of phenolic compounds. Here, we used a chemoinformatics approach to computationally identify endogenous ligands and assign putative biomolecular targets to oleacein, one of the most abundant secoiridoids in EVOO. Using a structure-based virtual profiling software tool and reference databases containing more than 9000 binding sites protein cavities, we identified 996 putative oleacein targets involving more than 700 proteins. We subsequently identified the high-level functions of oleacein in terms of biomolecular interactions, signaling pathways, and protein-protein interaction (PPI) networks. Delineation of the oleacein target landscape revealed that the most significant modules affected by oleacein were associated with metabolic processes (e.g., glucose and lipid metabolism) and chromatin-modifying enzymatic activities (i.e., histone post-translational modifications). We experimentally confirmed that, in a low-micromolar physiological range (<20 µmol/l), oleacein was capable of inhibiting the catalytic activities of predicted metabolic and epigenetic targets including nicotinamide N-methyltransferase, ATP-citrate lyase, lysine-specific demethylase 6A, and N-methyltransferase 4. Our computational de-orphanization of oleacein provides new mechanisms through which EVOO biophenols might operate as chemical prototypes capable of modulating the biologic machinery of healthy aging.


Assuntos
Aldeídos/metabolismo , Fenóis/metabolismo , Proteômica/métodos , ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/metabolismo , Aldeídos/química , Domínio Catalítico , Ensaios Enzimáticos , Epigenômica/métodos , Ontologia Genética/estatística & dados numéricos , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Humanos , Informática/métodos , Metiltransferases/química , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Nicotinamida N-Metiltransferase/química , Nicotinamida N-Metiltransferase/metabolismo , Olea/química , Azeite de Oliva/química , Fenóis/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Software
3.
Phytochemistry ; 164: 50-59, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078779

RESUMO

Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is synthesized by cinnamic acid methyltransferase (CAMT) that belongs to the SABATH family. While rarely reported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were designated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins, which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates. Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be 50.5 µM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and one flowering plant CAMT (ObCCMT1) in complex with (E)-cinnamic acid and salicylic acid were built, which provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis, CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes and flowering plants originated through convergent evolution.


Assuntos
Cinamatos/metabolismo , Hepatófitas/metabolismo , Metiltransferases/metabolismo , Cinamatos/química , Relação Dose-Resposta a Droga , Hepatófitas/química , Metiltransferases/química , Estrutura Molecular , Relação Estrutura-Atividade
4.
Int J Mol Sci ; 20(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934718

RESUMO

Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.


Assuntos
Apiaceae/metabolismo , Vias Biossintéticas , Cumarínicos/metabolismo , Sequência de Aminoácidos , Apiaceae/química , Apiaceae/genética , Cumarínicos/química , Mineração de Dados , Evolução Molecular , Furocumarinas/química , Furocumarinas/metabolismo , Duplicação Gênica , Heterozigoto , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/análise , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , Análise de Sequência de RNA , Especificidade por Substrato , Transcriptoma/genética , Umbeliferonas/química , Umbeliferonas/metabolismo
5.
Enzyme Microb Technol ; 125: 1-5, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885319

RESUMO

O-Methylation of N-acetylserotonin (NAS) has been identified as the bottleneck in melatonin biosynthesis pathway. In the present paper, caffeic acid O-methyltransferase from Arabidopsis thaliana (AtCOMT) was engineered by rational design to improve its catalytic efficiency in conversion of NAS to melatonin. Based on the notable difference in the terminal structure of caffeic acid and NAS, mutants were designed to strengthen the interactions between the substrate binding pocket of the enzyme and the terminal structure of the unnatural substrate NAS. The final triple mutant (C296F-Q310L-V314T) showed 9.5-fold activity improvement in O-methylation of NAS. Molecular dynamics simulations and binding free energy analysis attributed the increased activity to the higher affinity between the substrate terminal structure and AtCOMT, resulting from the introduction of NH⋯π interaction by Phe296 substitution, hydrophobic interaction by Thr314 substitution and elimination of electrostatic repulsion by substitution of Gln310 with Leu310. This work provides hints for O-methyltransferase engineering and meanwhile lays foundation for biotechnological production of melatonin.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Engenharia de Proteínas , Serotonina/análogos & derivados , Substituição de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Sítios de Ligação , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Catálise , Melatonina/biossíntese , Melatonina/metabolismo , Metiltransferases/genética , Simulação de Dinâmica Molecular , Estrutura Molecular , Serotonina/química , Serotonina/metabolismo , Relação Estrutura-Atividade , Termodinâmica
6.
Genes Dev ; 33(11-12): 620-625, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30923167

RESUMO

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.


Assuntos
Histonas/química , Histonas/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Arginina/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metilação , Modelos Moleculares , Estabilidade Proteica , Estrutura Secundária de Proteína , Ubiquitina/metabolismo , Ubiquitinação
7.
Org Lett ; 21(7): 2322-2325, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30855966

RESUMO

The biosynthetic gene cluster of antitumor antibiotic LL-D49194α1 (LLD) was identified and comparatively analyzed with that of trioxacarcins. The tailoring genes encoding glycosyltransferase, methyltransferase and cytochrome P450 were systematically deleted, which led to the discovery of eight compounds from the mutants. Preliminary pharmaceutical evaluation revealed two intermediates exhibiting higher cytotoxicity, stability and solubility. These results highlighted the modification pathway for LLD biosynthesis, and provided highly potent, structurally simplified "unnatural" natural products with improved druggability.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Metiltransferases/metabolismo , Antibióticos Antineoplásicos/química , Produtos Biológicos/química , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/química , Metiltransferases/química , Estrutura Molecular , Família Multigênica
8.
J Biochem ; 166(2): 139-147, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30828715

RESUMO

The lipids containing cyclopropane-fatty-acid (CFA) protect bacteria from adverse conditions such as acidity, freeze-drying desiccation and exposure to pollutants. CFA is synthesized when cyclopropane-fatty-acyl-phospholipid synthase (CFA synthase, CFAS) transfers a methylene group from S-adenosylmethionine (SAM) across the cis double bonds of unsaturated fatty acyl chains. Here, we reported a 2.7-Å crystal structure of CFAS from Lactobacillus acidophilus. The enzyme is composed of N- and C-terminal domain, which belong to the sterol carrier protein and methyltransferase superfamily, respectively. A phospholipid in the substrate binding site and a bicarbonate ion (BCI) acting as a general base in the active site were discovered. To elucidate the mechanism, a docking experiment using CFAS from L. acidophilus and SAM was carried out. The analysis of this structure demonstrated that three groups, the carbons from the substrate, the BCI and the methyl of S(CHn)3 group, were close enough to form a cyclopropane ring with the help of amino acids in the active site. Therefore, the structure supports the hypothesis that CFAS from L. acidophilus catalyzes methyl transfer via a carbocation mechanism. These findings provide a structural basis to more deeply understand enzymatic cyclopropanation.


Assuntos
Lactobacillus acidophilus/enzimologia , Metiltransferases/metabolismo , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Escherichia coli/citologia , Escherichia coli/metabolismo , Cinética , Lactobacillus acidophilus/metabolismo , Metiltransferases/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Fosfolipídeos/química , Alinhamento de Sequência
10.
Phytochemistry ; 159: 90-101, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30605853

RESUMO

The main polysaccharide of the gel present in the leaves of or Aloe vera Burm.F., (Aloe barbadensis Miller) a xerophytic crassulacean acid metabolism (CAM) plant, is an acetylated glucomannan named acemannan. This polysaccharide is responsible for the succulence of the plant, helping it to retain water. In this study we determined using polysaccharide analysis by carbohydrate gel electrophoresis (PACE) that the acemannan is a glucomannan without galactose side branches. We also investigated the expression of the gene responsible for acemannan backbone synthesis, encoding a glucomannan mannosyltransferase (GMMT, EC 2.4.1.32), since there are no previous reports on GMMT expression under water stress in general and specifically in Aloe vera. It was found by in silico analyses that the GMMT gene belongs to the cellulose synthase-like A type-9 (CSLA9) subfamily. Using RT-qPCR it was found that the expression of GMMT increased significantly in Aloe vera plants subjected to water stress. This expression correlates with an increase of endogenous ABA levels, suggesting that the gene expression could be regulated by ABA. To corroborate this hypothesis, exogenous ABA was applied to non-water-stressed plants, resulting in a significant increase of GMMT expression after 48 h of ABA treatment.


Assuntos
Ácido Abscísico/farmacologia , Aloe/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Mananas/metabolismo , Metiltransferases/genética , Estresse Fisiológico , Água/metabolismo , Aloe/enzimologia , Aloe/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , DNA Complementar/genética , Secas , Eletroforese em Gel de Amido/métodos , Cromatografia Gasosa-Espectrometria de Massas , Metiltransferases/química , Metiltransferases/metabolismo , Homologia de Sequência de Aminoácidos
11.
Phytochemistry ; 159: 190-198, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30634081

RESUMO

Previously it has been shown that the caffeoyl coenzyme A O-methyltransferase (CCoAOMT) type enzyme PaF6OMT, synthesized by the liverwort Plagiochasma appendiculatum Lehm. & Lindenb., (Aytoniaceae), interacts preferentially with 6-OH flavones. To clarify the biochemistry and evolution of liverwort OMTs, a comparison was made between the nucleotide sequence and biological activity of PaF6OMT and those of three of its homologs MpOMT1 (from Marchantia paleacea Bertol., (Marchantiaceae)), MeOMT1 (Marchantia emarginata Reinw et al., (Marchantiaceae)) and HmOMT1 (Haplomitrium mnioides (Lindb.) Schust., (Haplomitriaceae)). The four genes shared >60% level of sequence identity with one another but a <20% level of similarity with typical CCoAOMT or CCoAOMT-like sequences; they clustered with genes encoding animal catechol methyltransferases. The recombinant OMTs recognized phenylpropanoids, flavonoids and coumarins as substrates, but not catechol. MpOMT1 and PaF6OMT exhibited some differences with respect to their substrate preference, and the key residues underlying this preference were identified using site-directed mutagenesis. The co-expression of MpOMT1 and the Arabidopsis thaliana gene encoding S-adenosyl-L-methionine synthase in Escherichia coli was shown to be an effective means of enhancing the production of the pharmacologically active compounds scopoletin and oroxylin A. Liverwort OMTs are thought likely to represent an ancestral out-group of bona fide higher plant CCoAOMTs in evolution and have the potential to be exploited for the production of methylated flavones and coumarins.


Assuntos
Hepatófitas/enzimologia , Metiltransferases/metabolismo , Sequência de Aminoácidos , Catálise , Proliferação de Células/efeitos dos fármacos , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Genes de Plantas , Hepatófitas/classificação , Hepatófitas/genética , Metiltransferases/química , Metiltransferases/genética , Filogenia , Escopoletina/isolamento & purificação , Escopoletina/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato
12.
Org Biomol Chem ; 17(5): 1169-1175, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30644493

RESUMO

The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M3S domain from TioS(A3aM3SA3b) between the a8-a9 region of a mono-interrupted A domain, TioN(AaMNAb), that already contained a side chain methylating MN domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AMNAM3SA) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.


Assuntos
Monofosfato de Adenosina/química , Metiltransferases/metabolismo , Peptídeo Sintases/metabolismo , Engenharia de Proteínas , Aminoácidos/metabolismo , Catálise , Metilação , Metiltransferases/química , Metiltransferases/isolamento & purificação , Peptídeo Sintases/química , Peptídeo Sintases/isolamento & purificação , Peptídeos/química , Domínios Proteicos , Radiometria , Especificidade por Substrato , Espectrometria de Massas em Tandem
13.
Biochim Biophys Acta Gen Subj ; 1863(1): 182-190, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30308221

RESUMO

BACKGROUND: Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown. METHODS: To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and 77Se NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations. RESULTS: Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by 77Se NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy. CONCLUSIONS: The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner. GENERAL SIGNIFICANCE: Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid.


Assuntos
Espectroscopia de Ressonância Magnética , Metiltransferases/química , Selênio/química , Selenocisteína/química , Catálise , Domínio Catalítico , Humanos , Cinética , Metilação , Conformação Molecular , Simulação de Acoplamento Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Selenocisteína/análogos & derivados
14.
Org Biomol Chem ; 17(3): 477-481, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30565634

RESUMO

Toxoflavin (1), fervenulin (2), and reumycin (3), known to be produced by plant pathogen Burkholderia glumae BGR1, are structurally related 7-azapteridine antibiotics. Previous biosynthetic studies revealed that N-methyltransferase ToxA from B. glumae BGR1 catalyzed the sequential methylation at N6 and N1 in pyrimido[5,4-e]-as-triazine-5,7(6H,8H)-dione (4) to generate 1. However, the N8 methylation of 4 in the biosynthesis of fervenulin remains unclear. To explore the N-methyltransferases required for the biosynthesis of 1 and 2, we identified and characterized the fervenulin and toxoflavin biosynthetic gene clusters in S. hiroshimensis ATCC53615. On the basis of the structures of intermediates accumulated from the four N-methyltransferase gene inactivation mutants and systematic enzymatic methylation reactions, the tailoring steps for the methylation order in the biosynthesis of 1 and 2 were proposed. The N-methylation order and routes for the biosynthesis of fervenulin and toxoflavin in S. hiroshimensis are more complex and represent an obvious departure from those in B. glumae BGR1.


Assuntos
Metiltransferases/metabolismo , Pirimidinonas/metabolismo , Streptomyces/metabolismo , Triazinas/metabolismo , Biocatálise , Relação Dose-Resposta a Droga , Metiltransferases/química , Estrutura Molecular , Pirimidinonas/química , Streptomyces/química , Streptomyces/enzimologia , Relação Estrutura-Atividade , Triazinas/química
15.
Biochemistry ; 58(6): 665-678, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30525512

RESUMO

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet)-dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an SN2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.


Assuntos
Proteínas de Bactérias/química , Metiltransferases/química , Peptídeo Sintases/química , Fenóis/química , Tiazóis/química , Proteínas de Bactérias/isolamento & purificação , Catálise , Catecol O-Metiltransferase/química , Escherichia coli/genética , Cinética , Methanocaldococcus/enzimologia , Metionina Adenosiltransferase/química , Metionina Adenosiltransferase/isolamento & purificação , Metilação , Metiltransferases/isolamento & purificação , Peptídeo Sintases/isolamento & purificação , Fenóis/síntese química , Domínios Proteicos , Pseudomonas aeruginosa/enzimologia , S-Adenosilmetionina/análogos & derivados , Tiazóis/síntese química
17.
Biochemistry ; 57(50): 6827-6837, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30525509

RESUMO

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.


Assuntos
Aminoglicosídeos/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Micromonospora/enzimologia , Oxigenases/química , Oxigenases/metabolismo , Sequência de Aminoácidos , Aminoglicosídeos/química , Proteínas de Bactérias/genética , Vias Biossintéticas , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Fusão Gênica , Genes Bacterianos , Metiltransferases/genética , Micromonospora/genética , Modelos Moleculares , Oxigenases/genética , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de Aminoácidos
18.
Chem Commun (Camb) ; 54(90): 12718-12721, 2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30357150

RESUMO

Supplemented with synthetic surrogates of their natural cosubstrate S-adenosyl-l-methione (AdoMet), methyltransferases represent a powerful toolbox for the functionalization of biomolecules. By employing novel cosubstrate derivatives in combination with protein engineering, we show that this chemo-enzymatic method can be used to introduce photolabile protecting groups into DNA even in the presence of AdoMet. This approach enables optochemical control of gene expression in a straight-forward manner and we have termed it reversible methyltransferase directed transfer of photoactivatable groups (re-mTAG).


Assuntos
DNA/química , DNA/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/genética , Modelos Moleculares , Estrutura Molecular , Fotólise , Engenharia de Proteínas , Especificidade por Substrato
19.
Org Lett ; 20(17): 5427-5430, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30141637

RESUMO

Ovothiols are thiolhistidine derivatives. The first step of ovothiol biosynthesis is OvoA-catalyzed oxidative coupling between histidine and cysteine. In this report, the remaining steps of ovothiol A biosynthesis were reconstituted in vitro. ETA_14770 (OvoB) was reported as a PLP-dependent sulfoxide lyase, responsible for mercaptohistidine production. OvoA was found to be a bifunctional enzyme, which mediates both oxidative C-S bond formation and methylation of mercaptohistidine to afford ovothiol A. Besides reconstituting the whole biosynthetic pathway, two unique features proposed in the literature were also examined: a potential cysteine-recycling mechanism of the C-S lyase (OvoB) and the selectivity of the π- N methyltransferase.


Assuntos
Liases/metabolismo , Metilistidinas/metabolismo , Metiltransferases/metabolismo , Liases/química , Metilistidinas/química , Metiltransferases/química , Modelos Moleculares , Conformação Proteica
20.
Molecules ; 23(7)2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018257

RESUMO

Sterol 14α-demethylase (SDM) is essential for sterol biosynthesis and is the primary molecular target for clinical and agricultural antifungals. SDM has been demonstrated to be a valid drug target for antiprotozoal therapies, and much research has been focused on using SDM inhibitors to treat neglected tropical diseases such as human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis. Sterol C24-methyltransferase (24-SMT) introduces the C24-methyl group of ergosterol and is an enzyme found in pathogenic fungi and protozoa but is absent from animals. This difference in sterol metabolism has the potential to be exploited in the development of selective drugs that specifically target 24-SMT of invasive fungi or protozoa without adversely affecting the human or animal host. The synthesis and biological activity of SDM and 24-SMT inhibitors are reviewed herein.


Assuntos
Inibidores de 14-alfa Desmetilase , Proteínas Fúngicas , Metiltransferases , Micoses , Infecções por Protozoários , Proteínas de Protozoários , Esterol 14-Desmetilase , Inibidores de 14-alfa Desmetilase/síntese química , Inibidores de 14-alfa Desmetilase/química , Inibidores de 14-alfa Desmetilase/uso terapêutico , Animais , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/química , Metiltransferases/metabolismo , Micoses/tratamento farmacológico , Micoses/enzimologia , Infecções por Protozoários/tratamento farmacológico , Infecções por Protozoários/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/metabolismo
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