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1.
Chemistry ; 26(2): 454-463, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31603264

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t1/2 =6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-CuI reacts with sub-equimolar amounts of H2 O2 . UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub-equimolar amounts of H2 O2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems.


Assuntos
Cobre/química , Oxigenases de Função Mista/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/química , Cinética , Oxigenases de Função Mista/química , Oxirredução , Thermoascus/enzimologia
2.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140297, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672609

RESUMO

Two forms of C1/C4-oxidizing lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum (Talaromyces verruculosus) homologously expressed in P. verruculosum B1-537 auxotrophic strain were isolated in a homogeneous state using two-stage chromatography. The PvLPMO9A-hm form represented a full-size enzyme encoded by the intact lpmo1 gene, while the PvLPMO9A-lm was a truncated enzyme variant consisting of a conserved catalytic core of AA9 family LPMOs and lacking a C-terminal extra peptide sequence that is present in PvLPMO9A-hm. The N-terminal histidine was partially methylated in both enzymes. Most of properties of PvLPMO9A-hm and PvLPMO9A-lm, such as specific activities determined using the 2,6-dimethoxyphenol/H2O2 assay, pH-optima of activity observed at pH 7.5, synergistic effects exhibited with purified cellobiohydrolase I (Cel7A) and/or endoglucanase II (Cel5A) from P. verruculosum in hydrolysis of Avicel and milled aspen wood, were also very similar, except for the higher PvLPMO9A-hm thermostability studied using differential scanning calorimetry (DSC). The DSC profile for the PvLPMO9A-hm holoenzyme demonstrated two overlapping peaks (with maxima at 56.3 and 59.6 °C) due to the presence of two unfolding protein domains, while the PvLPMO9A-lm DSC profile represented one peak with maximum at 48.1 °C. After removing the active site copper with EDTA, the PvLPMO9A-hm and PvLPMO9A-lm melting temperatures decreased by ~10-11 and ~1 °C, respectively. These data show that both active site copper and C-terminal domain present in the PvLPMO9A-hm protect the enzyme from thermal unfolding, while the stabilizing effect of metal is much less pronounced in the truncated PvLPMO9A-lm form.


Assuntos
Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Penicillium/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Celulases/química , Celulose/química , Cobre/química , Estabilidade Enzimática , Proteínas Fúngicas/genética , Oxigenases de Função Mista/genética , Domínios Proteicos
3.
Nat Commun ; 10(1): 4910, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659163

RESUMO

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Membrana/química , Oxigenases de Função Mista/química , Proteínas Musculares/química , Sequência de Aminoácidos , Asparagina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Domínio Catalítico , Cristalografia , Dissulfetos/química , Dissulfetos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Conformação Proteica
4.
Int J Mol Sci ; 20(18)2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533304

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes in both the natural carbon cycle and the biorefinery industry. Understanding the molecular basis of LPMOs acting on polysaccharide substrates is helpful for improving industrial cellulase cocktails. Here we analyzed the sequences, structures, and substrate binding modes of LPMOs to uncover the factors that influence substrate specificity and regioselectivity. Our results showed that the different compositions of a motif located on L2 affect the electrostatic potentials of substrate binding surfaces, which in turn affect substrate specificities of AA10 LPMOs. A conserved Asn at a distance of 7 Å from the active center Cu might, together with the conserved Ser immediately before the second catalytic His, determine the localization of LPMOs on substrate, and thus contribute to C4-oxidizing regioselectivity. The findings in this work provide an insight into the molecular basis of substrate specificity and regioselectivity of LPMOs.


Assuntos
Sequência de Aminoácidos , Oxigenases de Função Mista/química , Modelos Moleculares , Sítios de Ligação , Oxigenases de Função Mista/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Nat Commun ; 10(1): 3611, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399587

RESUMO

The formicamycin biosynthetic gene cluster encodes two groups of type 2 polyketide antibiotics: the formicamycins and their biosynthetic precursors the fasamycins, both of which have activity against methicillin-resistant Staphylococcus aureus. Here, we report the formicapyridines which are encoded by the same gene cluster and are structurally and biosynthetically related to the fasamycins and formicamycins but comprise a rare pyridine moiety. These compounds are trace-level metabolites formed by derailment of the major biosynthetic pathway. Inspired by evolutionary logic we show that rational mutation of a single gene in the biosynthetic gene cluster encoding an antibiotic biosynthesis monooxygenase (ABM) superfamily protein leads to a significant increase both in total formicapyridine production and their enrichment relative to the fasamycins/formicamycins. Our observations broaden the polyketide biosynthetic landscape and identify a non-catalytic role for ABM superfamily proteins in type II polyketide synthase assemblages for maintaining biosynthetic pathway fidelity.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/química , Oxigenases de Função Mista/química , Policetídeos/metabolismo , Domínios Proteicos/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Engenharia Metabólica , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxigenases de Função Mista/genética , Família Multigênica , Mutação , Metabolismo Secundário , Streptomyces/genética , Streptomyces/metabolismo
6.
Protein Pept Lett ; 26(5): 377-385, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237199

RESUMO

BACKGROUND: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet. OBJECTIVE: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO. METHODS: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail. RESULTS: LPMO was highly induced in complex biomass like sugarcane bagasse (SEB), Avicel® PH-101, and CM-cellulose. The LPMO gene encoded a protein comprising 250 amino acids, without a CBM domain. After protein purification, the AfAA9_B molecular mass estimated by SDSPAGE was 35 kDa. The purified protein specific peroxidase activity was 8.33 ± 1.9 U g-1. Upon addition to Celluclast 1.5L, Avicel® PH-101 and SEB hydrolysis increased by 18% and 22%, respectively. CONCLUSION: A. fumigatus LPMO is a promising candidate to enhance the currently available enzymatic cocktail and can therefore be used in second-generation ethanol production.


Assuntos
Aspergillus fumigatus/enzimologia , Celulose/química , Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Polissacarídeos/química , Saccharum/química , Biomassa , Escherichia coli/genética , Etanol/química , Proteínas Fúngicas/genética , Hidrólise , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
7.
Methods Enzymol ; 620: 399-422, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072495

RESUMO

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems.


Assuntos
Ensaios Enzimáticos/métodos , FMN Redutase/química , Oxigenases de Função Mista/química , Flavinas/química , Cinética , Oxirredução , Ligação Proteica , Especificidade por Substrato
8.
Carbohydr Res ; 478: 46-53, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054382

RESUMO

A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be ~27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan.


Assuntos
Ascomicetos/enzimologia , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Biocatálise , Configuração de Carboidratos , Oxigenases de Função Mista/química , Simulação de Acoplamento Molecular , Polissacarídeos/química
9.
Chemistry ; 25(48): 11257-11268, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31131927

RESUMO

Selective oxidation reactions of organic compounds with dioxygen using molecular copper complexes are of relevance to synthetic chemistry as well as enzymatic reactivity. In the enzyme peptidylglycine α-hydroxylating monooxygenase (PHM), the hydroxylating activity towards aliphatic substrates arises from the cooperative effect between two copper atoms, but the detailed mechanism has yet to be fully clarified. Herein, we report on a model complex showing hydroxylation of an aliphatic ligand initiated by dioxygen. According to DFT calculations, the proton-coupled electron-transfer (PCET) process leading to ligand hydroxylation in this complex benefits from cooperative effects between the two copper atoms. While one copper atom is responsible for dioxygen binding and activation, the other stabilizes the product of intramolecular PCET by copper-ligand charge transfer. The results of this work might pave the way for the directed utilization of cooperative effects in oxidation reactions.


Assuntos
Complexos de Coordenação/química , Cobre/química , Guanidinas/química , Oxigênio/metabolismo , Sítios de Ligação , Teoria da Densidade Funcional , Transporte de Elétrons , Hidroxilação , Ligantes , Oxigenases de Função Mista/química , Modelos Moleculares , Complexos Multienzimáticos/química , Oxirredução
10.
Oncol Res Treat ; 42(6): 309-318, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31055566

RESUMO

DNA methylation plays significant roles in a variety of biological and pathological processes including mammalian development, genomic imprinting, retrotransposon silencing, and X-chromosome inactivation. Recent discoveries indicated that ten-eleven translocation (TET) family of dioxygenases can convert 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). The TET family includes three members: TET1, TET2, and TET3. With increasing evidence, more and more biological and pathological processes in which 5-hmC and TET family serve unparalleled biological roles are noticed, for example, DNA demethylation and transcriptional regulation of different target genes, which are involved in many human diseases, especially hematologic malignancies, resembling chronic myelomonocytic leukemia, myelodysplastic syndromes, and so on. In this review, we focus on the diverse functions of TET family and the novel epigenetic marks, 5-mC and 5-hmC, in hematologic malignancies. This review will provide valuable insights into the potential targets of hematologic malignancies. Further understanding of the normal and pathological functions of TET family may provide new methods to develop novel epigenetic therapies for treating hematologic malignancies.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Epigênese Genética , Neoplasias Hematológicas/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Metilação de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dioxigenases/química , Dioxigenases/genética , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Mutação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética
11.
PLoS One ; 14(4): e0216134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31034531

RESUMO

The asparaginyl hydroxylase, Factor Inhibiting HIF (FIH), is a cellular dioxygenase. Originally identified as oxygen sensor in the cellular response to hypoxia, where FIH acts as a repressor of the hypoxia inducible transcription factor alpha (HIF-α) proteins through asparaginyl hydroxylation, FIH also hydroxylates many proteins that contain ankyrin repeat domains (ARDs). Given FIH's promiscuity and the unclear functional effects of ARD hydroxylation, the biological relevance of HIF-α and ARD hydroxylation remains uncertain. Here, we have employed evolutionary and enzymatic analyses of FIH, and both HIF-α and ARD-containing substrates, in a broad range of metazoa to better understand their conservation and functional importance. Utilising Tribolium castaneum and Acropora millepora, we provide evidence that FIH from both species are able to hydroxylate HIF-α proteins, supporting conservation of this function beyond vertebrates. We further demonstrate that T. castaneum and A. millepora FIH homologs can also hydroxylate specific ARD proteins. Significantly, FIH is also conserved in several species with inefficiently-targeted or absent HIF, supporting the hypothesis of important HIF-independent functions for FIH. Overall, these data show that while oxygen-dependent HIF-α hydroxylation by FIH is highly conserved in many species, HIF-independent roles for FIH have evolved in others.


Assuntos
Antozoários/enzimologia , Sequência Conservada , Oxigenases de Função Mista/metabolismo , Tribolium/enzimologia , Sequência de Aminoácidos , Animais , Repetição de Anquirina , Hipóxia Celular , Evolução Molecular , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigenases de Função Mista/química , Ligação Proteica , Transdução de Sinais , Especificidade por Substrato
12.
Acta Crystallogr D Struct Biol ; 75(Pt 4): 368-380, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30988254

RESUMO

Neutron crystallography is a powerful method to determine the positions of H atoms in macromolecular structures. However, it is sometimes hard to judge what would constitute a chemically reasonable model, and the geometry of H atoms depends more on the surroundings (for example the formation of hydrogen bonds) than heavy atoms, so that the empirical geometry information for the H atoms used to supplement the experimental data is often less accurate. These problems may be reduced by using quantum-mechanical calculations. A method has therefore been developed to combine quantum-mechanical calculations with joint crystallographic refinement against X-ray and neutron data. A first validation of this method is provided by re-refining the structure of the galectin-3 carbohydrate-recognition domain in complex with lactose. The geometry is improved, in particular for water molecules, for which the method leads to better-resolved hydrogen-bonding interactions. The method has also been applied to the active copper site of lytic polysaccharide monooxygenase and shows that the protonation state of the amino-terminal histidine residue can be determined.


Assuntos
Cristalografia por Raios X/métodos , Galectina 3/química , Oxigenases de Função Mista/química , Nêutrons , Polissacarídeos/química , Conformação Proteica , Domínio Catalítico , Humanos , Modelos Moleculares , Estrutura Molecular , Teoria Quântica
13.
Int J Mol Sci ; 20(7)2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30986901

RESUMO

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme's activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.


Assuntos
Evolução Molecular Direcionada , Enzimas Imobilizadas/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Animais , Bovinos , Fluoresceína-5-Isotiocianato/metabolismo , Mutação/genética , Engenharia de Proteínas , Saccharomyces cerevisiae
14.
BMC Evol Biol ; 19(1): 76, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30866798

RESUMO

BACKGROUND: Unspecific peroxygenases (UPO) (EC 1.11.2.1) represent an intriguing oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity. With over 300 identified substrates, UPOs catalyze numerous oxidations including 1- or 2- electron oxygenation, selective oxyfunctionalizations, which make them most significant in organic syntheses and potentially attractive as industrial biocatalysts. There are very few UPOs available with distinct properties, notably, MroUPO which shows behavior ranging between UPO and another heme-thiolate peroxidase, called Chloroperoxidase (CPO). It prompted us to search for more UPOs in fungal kingdom which led us to studying their relationship with CPO. RESULTS: In this study, we searched for novel UPOs in more than 800 fungal genomes and found 113 putative UPO-encoding sequences distributed in 35 different fungal species (or strains), amongst which single sequence per species were subjected to phylogeny study along with CPOs. Our phylogenetic study show that the UPOs are distributed in Basidiomycota and Ascomycota phyla of fungi. The sequence analysis helped to classify the UPOs into five distinct subfamilies: classic AaeUPO and four new subfamilies with potential new traits. We have also shown that each of these five subfamilies (supported by) have their own signature motifs. Surprisingly, some of the CPOs appeared to be a type of UPOs indicating that they were previously identified incorrectly. Selection pressure was observed on important motifs in UPOs which could have driven their functional divergence. Furthermore, the sites having different evolutionary rates caused by the functional divergence were also identified on some motifs along with the other relevant amino acid residues. Finally, we predicted critical amino acids responsible for the functional divergence in the UPOs and identified some sequence differences among UPOs, CPOs, and MroUPO to predict it's ranging behavior. CONCLUSION: This study discovers new UPOs, provides a glimpse of their evolution from CPOs, and presents new insight on their functional divergence. We present a new classification of UPOs and shed new light on its phylogenetics. These different UPOs may exhibit a wide range of characteristics and specificities which may help in various fields of synthetic chemistry and industrial biocatalysts, and may as well lead to an advancement towards the understanding of physiological role of UPOs in fungi.


Assuntos
Evolução Molecular , Oxigenases de Função Mista/metabolismo , Família Multigênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Ascomicetos/enzimologia , Basidiomycota/enzimologia , Sequência Conservada , Variação Genética , Funções Verossimilhança , Oxigenases de Função Mista/química , Filogenia , Seleção Genética
15.
Int J Mol Sci ; 20(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925699

RESUMO

High population density alters insect prophylactic immunity, with density-dependent prophylaxis (DDP) being reported in many polyphonic insects. However, the molecular mechanism for DDP remains unclear. In current study, the role of tyramine ß-hydroxylase (Tßh) in the immune response of M. separata larvae that were subject to different rearing densities conditions was investigated. The tyramine ß-hydroxylase activity of larvae from high density treatments (10 and 30 larvae per jar) was significantly higher than that of the larvae from low density treatments (one, two, and five larvae/jar). A tyramine ß-hydroxylase (designated MsTßh) containing a 1779 bp open reading frame was identified. Multiple sequence alignment and phylogenetic analysis indicated that MsTßh was orthologous to the Tßh that was found in other lepidopterans. Elevated MsTßh expression was observed in larvae under high density (10 larvae per jar). Silencing MsTßh expression by the injection of dsRNA in larvae from the high density treatment produced a 25.1% reduction in octopamine levels, while at the same time, there was a significant decrease in phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival against Beauveria infection 56.6%, 88.5%, 82.0%, and 55.8%, respectively, when compared to control larvae. Our findings provide the first insights into how MsTßh mediates the octopamine level, which in turn modulates the immune response of larvae under different population densities.


Assuntos
Proteínas de Insetos/imunologia , Oxigenases de Função Mista/imunologia , Mariposas/imunologia , Sequência de Aminoácidos , Animais , Beauveria/imunologia , Imunidade , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/química , Larva/genética , Larva/imunologia , Larva/microbiologia , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Mariposas/química , Mariposas/genética , Mariposas/microbiologia , Filogenia , Alinhamento de Sequência
16.
Biochem J ; 476(7): 1109-1119, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30898847

RESUMO

The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2S)-N ε-trimethyllysine to (2S,3S)-3-hydroxy-N ε-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate and (2S)-N ε-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2S)-N ε-trimethyllysine to (2S,3S)-3-hydroxy-N ε-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242-D244-H389 residues, R391-R398 involved in 2OG binding and several residues (D231, N334 and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2S)-N ε-trimethyllysine.


Assuntos
Oxigenases de Função Mista/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Carnitina/biossíntese , Domínio Catalítico/genética , Humanos , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , gama-Butirobetaína Dioxigenase/química , gama-Butirobetaína Dioxigenase/genética , gama-Butirobetaína Dioxigenase/metabolismo
17.
Planta ; 249(6): 1987-1996, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30903269

RESUMO

MAIN CONCLUSION: Amino acid sequence and crystal structure analyses of Tma12, an insecticidal protein isolated from the fern Tectaria macrodonta, identify it as a carbohydrate-binding protein belonging to the AA10 family of lytic polysaccharide monooxygenases, and provide the first evidence of AA10 proteins in plants. Tma12, isolated from the fern Tectaria macrodonta, is a next-generation insecticidal protein. Transgenic cotton expressing Tma12 exhibits resistance against whitefly and viral diseases. Beside its insecticidal property, the structure and function of Tma12 are unknown. This limits understanding of the insecticidal mechanism of the protein and targeted improvement in its efficacy. Here we report the amino acid sequence analysis and the crystal structure of Tma12, suggesting that it is possibly a lytic polysaccharide monooxygenase (LPMO) of the AA10 family. Amino acid sequence of Tma12 shows 45% identity with a cellulolytic LPMO of Streptomyces coelicolor. The crystal structure of Tma12, obtained at 2.2 Å resolution, possesses all the major structural characteristics of AA10 LPMOs. A H2O2-based enzymatic assay also supports this finding. It is the first report of the occurrence of LPMO-like protein in a plant. The two facts that Tma12 possesses insecticidal activity and shows structural similarity with LPMOs collectively advocate exploration of microbial LPMOs for insecticidal potential.


Assuntos
Gleiquênias/enzimologia , Hemípteros/efeitos dos fármacos , Inseticidas/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Gleiquênias/química , Gleiquênias/genética , Gossypium/enzimologia , Gossypium/genética , Gossypium/fisiologia , Peróxido de Hidrogênio/metabolismo , Inseticidas/farmacologia , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência
18.
Org Lett ; 21(7): 2330-2334, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30900461

RESUMO

Ascochlorin is a medicinally important fungal meroterpenoid. Its biosynthetic pathway in Fusarium sp. was identified, and the stereoselective epoxidation of the farnesyl group by the multidomain, soluble P450 monooxygenase AscE and the subsequent formation of the unique timethylcyclohexanone ring by the membrane-bound cyclase AscF were investigated. Precursor-directed biosynthesis generated novel bromo-substituted derivatives, which exhibited potent cytotoxic activities. This study paves the way for the future metabolic engineering of medicinally important meroterpenoids for drug discovery.


Assuntos
Alcenos/química , Fungos/metabolismo , Fusarium/química , Oxigenases de Função Mista/metabolismo , Oxirredutases/metabolismo , Fenóis/química , Terpenos/química , Vias Biossintéticas , Oxigenases de Função Mista/química , Estrutura Molecular , Oxirredutases/química , Oxirredutases/isolamento & purificação , Terpenos/isolamento & purificação
19.
Biochem Biophys Res Commun ; 510(4): 601-605, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30739782

RESUMO

AlpK is an essential monooxygenase involved in the biosynthesis of kinamycin. It catalyzes the C5-hyfroxylattion of the crucial benzo[b]-fluorence intermediate in kinamycin synthesis. However, the structure and mechanism of AlpK is unclear. Here, we report the first structure of AlpK in complex with FAD. Our structure sheds light on the catalytic mechanism of AlpK.


Assuntos
Proteínas de Bactérias/química , Oxigenases de Função Mista/química , Streptomyces/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Quinonas/metabolismo , Alinhamento de Sequência , Streptomyces/química , Streptomyces/metabolismo
20.
J Agric Food Chem ; 67(8): 2245-2254, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30721044

RESUMO

Chlorpyrifos is one of the most used organophosphorus insecticides. It is commonly degraded to 3,5,6-trichloro-2-pyridinol (TCP), which is water-soluble and toxic. Bacteria can degrade chlorpyrifos and TCP, but the biodegradation mechanism has not been well-characterized. Recently isolated Cupriavidus nantongensis X1T can completely degrade 100 mg/L chlorpyrifos and 20 mg/L TCP with half-lives of 6 and 8 h, respectively. We annotated a complete gene cluster responsible for TCP degradation in recently sequenced strain X1T. Two key genes, tcpA and fre, were cloned from X1T and transferred and expressed in Escherichia coli BL21(DE3). Degradation of TCP by X1T whole cell was compared with that by the enzymes 2,4,6-trichlorophenol monooxygenase and NAD(P)H:flavin reductase expressed and purified from E. coli BL21(DE3). Novel metabolites of TCP were isolated and characterized, indicating stepwise dechlorination of TCP, which was confirmed by TCP disappearance, mass balance, and detection and formation kinetics of chloride ion from TCP.


Assuntos
Proteínas de Bactérias/química , Clorpirifos/metabolismo , Cupriavidus/enzimologia , FMN Redutase/química , Inseticidas/metabolismo , Oxigenases de Função Mista/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Clorpirifos/química , Cupriavidus/química , Cupriavidus/genética , Cupriavidus/metabolismo , FMN Redutase/genética , FMN Redutase/metabolismo , Halogenação , Inseticidas/química , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo
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