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1.
Appl Microbiol Biotechnol ; 108(1): 141, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38231240

RESUMO

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in eukaryotes that sense and transduce extracellular signals into cells. In Aspergillus oryzae, 16 canonical GPCR genes are identified and classified into nine classes based on the sequence similarity and proposed functions. Class VI GPCRs (AoGprK-1, AoGprK-2, and AoGprR in A. oryzae), unlike other GPCRs, feature a unique hybrid structure containing both the seven transmembrane (7-TM) and regulator of G-protein signaling (RGS) domains, which is not found in animal GPCRs. We report here that the mutants with double or triple deletion of class VI GPCR genes produced significantly increased number of sclerotia compared to the control strain when grown on agar plates. Interestingly, complementation analysis demonstrated that the expression of the RGS domain without the 7-TM domain is sufficient to restore the phenotype. In line with this, among the three Gα subunits in A. oryzae, AoGpaA, AoGpaB, and AoGanA, forced expression of GTPase-deficient mutants of either AoGpaA or AoGpaB caused an increase in the number of sclerotia formed, suggesting that RGS domains of class VI GPCRs are the negative regulators of these two GTPases. Finally, we measured the expression of velvet complex genes and sclerotia formation-related genes and found that the expression of velB was significantly increased in the multiple gene deletion mutants. Taken together, these results demonstrate that class VI GPCRs negatively regulate sclerotia formation through their GTPase-activating activity in the RGS domains. KEY POINTS: • Class VI GPCRs in A. oryzae regulate sclerotia formation in A. oryzae • RGS function of class VI GPCRs is responsible for regulation of sclerotia formation • Loss of class VI GPCRs resulted in increased expression of sclerotia-related genes.


Assuntos
Aspergillus oryzae , Animais , Aspergillus oryzae/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Ágar , GTP Fosfo-Hidrolases
2.
J Gen Appl Microbiol ; 68(6): 278-286, 2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-35858815

RESUMO

Glucuronoyl esterase (GE) is a promising agent for the delignification of plant biomass since it has been shown to cleave the linkage between xylan and lignin in vitro. In this study, we demonstrate that NcGE, a GE from Neurospora crassa, stimulates plant biomass degradation. In vitro, NcGE synergistically increased the release of reducing sugars from plant biomass when added together with cellulase or xylanase. In vivo, overexpression of NcGE in N. crassa resulted in an increase in xylanolytic activity. Consistently, elevated transcription of genes encoding the major plant biomass degrading-enzymes (PBDEs) was observed in the NcGE overexpression strain. Increased xylanolytic activity and transcription of PDBE genes were largely abolished when the transcription factors clr-1, clr-2, or xlr-1 were deleted. Interestingly, the expression of some PBDE genes was increased when the hydrolysate of plant biomass by NcGE was added to the culture medium. We propose that NcGE boosts the production of PBDEs through the activation of key transcription factors, which is presumably caused by NcGE-mediated generation of hypothetical inducer(s) from plant biomass.


Assuntos
Esterases , Neurospora crassa , Esterases/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Éteres Difenil Halogenados/metabolismo , Biomassa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
ACS Chem Biol ; 17(1): 207-216, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35000376

RESUMO

Ferrichromes are a family of fungal siderophores with cyclic hexapeptide structures. Most fungi produce one or two ferrichrome-type siderophores. Acremonium persicinum MF-347833 produces ferrichrome-like potent Trojan horse antifungal antibiotics ASP2397 and AS2488053, the aluminum- and iron-chelating forms of AS2488059, respectively. Here, we show by gene sequencing followed by gene deletion experiments that A. persicinum MF-347833 possesses two nonribosomal peptide synthetase genes responsible for AS2488059 and ferricrocin assembly. AS2488059 was produced under iron starvation conditions and excreted into the media to serve as a defense metabolite and probably an iron courier. In contrast, ferricrocin was produced under iron-replete conditions and retained inside the cells, likely serving as an iron-sequestering molecule. Notably, the phylogenetic analyses suggest the different evolutionary origin of AS2488059 from that of conventional ferrichrome-type siderophores. Harnessing two ferrichrome-type siderophores with distinct biological properties may give A. persicinum a competitive advantage for surviving the natural environment.


Assuntos
Acremonium/química , Complexos de Coordenação/metabolismo , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Peptídeos Cíclicos/metabolismo , Sideróforos/metabolismo , Complexos de Coordenação/química , Mineração de Dados , Ferricromo/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Peptídeos Cíclicos/química , Filogenia , Sideróforos/química
4.
Biosci Biotechnol Biochem ; 85(12): 2429-2433, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34643660

RESUMO

We found that the expression of genes involved in kojic acid (KA) biosynthesis, kojA, kojR, and kojT, was highly elevated in the Aspergillus oryzae autophagy-deficient mutants. In agreement, KA production was much increased in these mutants. Nuclear translocation of KojR, a transcription factor, was observed in the autophagy mutants before they were starved, explaining why KA production was boosted.


Assuntos
Aspergillus oryzae
5.
Int J Biol Macromol ; 169: 302-310, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33333093

RESUMO

Neurospora crassa possesses six putative xylanases and four putative xylosidases. qRT-PCR results showed that the expression of all these xylanolytic enzymes was induced by xylan. Except for two intracellular ß-xylosidases, others were shown to be secreted enzymes based on the localization analysis of EGFP-fusion proteins. Among them, GH10-1, GH10-2, GH11-1, and GH11-2 were successfully expressed and characterized as typical endo-ß-1,4-xylanases that hydrolyze the xylooligosaccharides with a polymeric degree not less than three or four. Strains deleted for either gh10-1, gh10-2, gh3-7, or gh3-8 displayed decreased growth in xylan and biomass media. Disruption of gh3-7 or gh43-1 resulted in enhanced-xylanolytic enzyme activity when cultivated in biomass medium. Collectively, these results suggest that xylooligosaccharides released by the actions of xylanases and xylosidases not only serve as the carbon sources to maintain the growth of N. crassa, but they also act as inducers to trigger the expression of hydrolytic enzymes in vivo.


Assuntos
Neurospora crassa/metabolismo , Xilanos/química , Xilanos/metabolismo , Biomassa , Metabolismo dos Carboidratos/fisiologia , Endo-1,4-beta-Xilanases/metabolismo , Glucuronatos/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Especificidade por Substrato , Xilosidases/química , Xilosidases/metabolismo
6.
Fungal Genet Biol ; 144: 103441, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32777385

RESUMO

To better understand the roles of genes involved in mannan degradation in filamentous fungi, in this study we searched, identified, and characterized one putative GH5 endo-ß-mannanase (GH5-7) and two putative GH2 mannan-degrading enzymes (GH2-1 and GH2-4) in Neurospora crassa. Real-time RT-PCR analyses showed that the expression levels of these genes were significantly up-regulated when the cells were grown in mannan-containing media where the induction level of gh5-7 was the highest. All three proteins were heterologously expressed and purified. GH5-7 displayed a substrate preference toward galactomannan by showing 10-times higher catalytic efficiency than to linear ß-mannan. In contrast, GH2-1 preferred short manno-oligosaccharides or ß-mannan as substrates. Compared to the wild type strain, the growth of Δgh5-7 and Δgh5-7Δgh2-4 mutants, but not Δgh2-1, Δgh2-4, and Δgh2-1Δgh2-4 mutants, was poor in the cultures containing glucomannan or galactomannan as the sole carbon source, suggesting that GH5-7 plays a critical role in the utilization of heteromannans in vivo. On the other hand, all the mutants showed significantly slow growth when grown in the medium containing linear ß-mannan. Collectively, these results indicate that N. crassa can utilize glucomannan and galactomannan without GH2-1 and GH2-4, but efficient degradation of ß-mannan requires a concerted action of three enzymes, GH5-7, GH2-1, and GH2-4.


Assuntos
Fungos/metabolismo , Mananas/metabolismo , Neurospora crassa/genética , beta-Manosidase/genética , Sequência de Aminoácidos/genética , Catálise , Fungos/genética , Hidrólise , Mananas/genética , Neurospora crassa/metabolismo , Oligossacarídeos/metabolismo , Especificidade por Substrato/genética
7.
J Microbiol Biotechnol ; 30(2): 296-305, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32120462

RESUMO

Tricholoma matsutake is an ectomycorrhizal fungus, related with the host of Pinus densiflora. Most of studies on T. matsutake have focused on mycelial growth, genes and genomics, phylogenetics, symbiosis, and immune activity of this strain. T. matsutake is known for its unique fragrance in Eastern Asia. The most major component of its scent is (R)-(-)-1-octen-3-ol and is biosynthesized from the substrate linoleic acid by the sequential reaction of lipoxygenase and peroxide lyase. Here, we report for the first time the biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake using the yeast Saccharomyces cerevisiae as a host. In this study, cDNA genes correlated with these reactions were cloned from T. matsutake, and expression studies of theses genes were carried out in the yeast Saccharomyces cerevisiae. The product of these genes expression study was carried out with Western blotting. The biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake in recombinant Saccharomyces cerevisiae was subsequently identified with GC-MS chromatography analysis. The biosynthesis of (R)-(-)-1-octen-3-ol with S. cerevisiae represents a significant step forward.


Assuntos
Aldeído Liases/genética , Sistema Enzimático do Citocromo P-450/genética , Expressão Gênica , Lipoxigenase/genética , Octanóis/metabolismo , Saccharomyces cerevisiae/metabolismo , Tricholoma/enzimologia , Tricholoma/genética , Clonagem Molecular , Fermentação , Isoenzimas , Proteínas Recombinantes , Temperatura , Transformação Genética
8.
Biochem Biophys Res Commun ; 518(4): 644-650, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31466721

RESUMO

Phospholipases A1 (PLA1s) catalyze the hydrolysis of sn-1 linkage in the glycerophospholipids, thereby releasing fatty acids and 2-acyl lysophospholipids. PLA1s are found in various organisms and tissues where they play diverse cellular functions, but their roles in filamentous fungi remain elusive. In this study we analyzed the enzymatic properties and physiological functions of two secretory PLA1s, PLA1-1 and its paralog PLA1-2, in the filamentous fungus Aspergillus oryzae. Although PLA1-1 and PLA1-2 share 49% amino acid sequence identity, they significantly differ in various aspects. While PLA1-1 displayed PLA1 activity to phosphatidylcholine and phosphatidylethanolamine, and degraded various phospholipids, PLA1-2 exhibited PLA1 activity only to phosphatidylglycerol. PLA1-1 was secreted to the culture medium, but PLA1-2 was not secreted and retained in the mycelium. Fluorescence microscopic observation of A. oryzae strains expressing EGFP-fused PLA1-1 and PLA1-2 demonstrated that they display overlapping but distinct cellular localization. A. oryzae mutants deleted for pla1-1 or pla1-2 grew normally, but the secreted phospholipase activity was significantly reduced in the Δpla1-1 strain. These data suggest that two sPLA1 enzymes are not redundant and play distinct cellular functions in A. oryzae.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/metabolismo , Fosfolipases A1/metabolismo , Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia de Fluorescência , Mutação , Micélio/enzimologia , Micélio/genética , Fosfolipases A1/genética , Fosfolipídeos/metabolismo
9.
Carbohydr Res ; 474: 1-7, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30665024

RESUMO

Symbionts in the gut of termites are expected to be large sources of enzymes involved in lignocellulose degradation, but their biotechnological potential has not been fully explored. In this study, we expressed, purified, and biochemically characterized a glycoside hydrolase family 11 xylanase, NtSymX11, from a symbiotic bacterium of the higher termite, Nasutitermes takasagoensis. NtSymX11 is a multimodular enzyme consisting of a catalytic domain and two tandem carbohydrate-binding modules (CBM36). The pH and temperature optima of NtSymX11 were pH 6.0 and 40 °C, respectively. By comparing the properties of full-length and truncated variants of NtSymX11, it was shown that CBM36 decreases the enzyme stability at acidic pH and high temperature. The main products from xylohexaose and various xylan substrates were X1-X3 xylooligosaccharides. Analysis of kinetic parameters indicated that NtSymX11 displays an outstanding catalytic performance when compared to other reported xylanases, and CBM36 enhances the activity by increasing the affinity to the substrate. Addition of Ca2+ boosted the activity of full-length enzyme, but not the truncated variant lacking the CBM, against the insoluble substrate, suggesting that CBM36 plays a role in the Ca2+-dependent increase of catalytic efficiency.


Assuntos
Bactérias/química , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Glucuronatos/metabolismo , Isópteros/microbiologia , Oligossacarídeos/metabolismo , Xilanos/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Cálcio/química , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucuronatos/química , Concentração de Íons de Hidrogênio , Intestinos/microbiologia , Cinética , Metagenoma , Oligossacarídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Simbiose/fisiologia , Temperatura , Xilanos/química
10.
Appl Microbiol Biotechnol ; 102(5): 2191-2201, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29332217

RESUMO

Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of D-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus. AfGE was heterologously expressed in Aspergillus oryzae, and the purified enzyme displayed the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. AfGE is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40-50 °C at pH 5. Molecular modeling and site-directed mutagenesis studies of AfGE demonstrated that Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring.


Assuntos
Aspergillus fumigatus/enzimologia , Esterases/metabolismo , Ésteres/metabolismo , Proteínas Fúngicas/química , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Estabilidade Enzimática , Esterases/química , Esterases/genética , Esterases/isolamento & purificação , Ésteres/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Ácido Glucurônico/metabolismo , Estrutura Molecular , Polissacarídeos/metabolismo , Especificidade por Substrato
11.
Appl Microbiol Biotechnol ; 102(4): 1737-1747, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29305697

RESUMO

Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two ß-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to ß-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp79) that stacks against the sugar ring at subsite - 5. The Km values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the kcat remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite - 5 has an important role in the recognition of the sugar ring in the substrate.


Assuntos
Isópteros/microbiologia , Mananas/metabolismo , Microbiota , beta-Manosidase/genética , beta-Manosidase/metabolismo , Animais , Clonagem Molecular , Galactose/análogos & derivados , Trato Gastrointestinal/microbiologia , Expressão Gênica , Modelos Moleculares , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , beta-Manosidase/química
12.
Enzyme Microb Technol ; 97: 104-113, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28010766

RESUMO

A ß-glucosidase (BG), PaBG1b, from the xylophagous cockroach Panesthia angustipennis spadica was heterologously expressed in the methylotrophic yeast Pichia pastoris, purified, and biochemically characterized. Post-translational modification and N-terminal sequencing analysis demonstrated that the expression product was comprised of two polypeptides with different N-terminal sequences, presumably due to the presence of lysine-arginine (KR) sequence in the putative mature region. Substrate specificity analysis showed that PaBG1b hydrolyzed a broad range of substrates including cellohexaose, with the preference for aryl ß-d-fucosyl linkage and laminaribiose. Although the glucose tolerance of PaBG1b was moderate (Ki=200.3±1.1mM), PaBG1b demonstrated high specific activity and catalytic efficiency towards cellobiose with Vmax and kcat/Km values of 436.7±6.3U/mg and 109.8mM-1s-1, respectively. In addition, PaBG1b was not inhibited by cellobiose up to the highest concentration tested (100mM). Collectively, our work demonstrates that PaBG1b is a potentially valuable BG for commercial bioethanol production from cellulose.


Assuntos
Celobiose/metabolismo , Baratas/enzimologia , Proteínas de Insetos/metabolismo , beta-Glucosidase/metabolismo , Animais , Biocombustíveis , Baratas/genética , Estabilidade Enzimática , Etanol/metabolismo , Genes de Insetos , Proteínas de Insetos/genética , Cinética , Pichia/enzimologia , Pichia/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , beta-Glucosidase/genética
13.
Biochem Biophys Res Commun ; 480(3): 302-308, 2016 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-27746179

RESUMO

In mammals, three types of intracellular phospholipase A1 (iPLA1) enzymes have been characterized and are thought to be involved in various cellular processes such as phospholipid metabolism, organelle biogenesis, and membrane trafficking. In this study we analyzed the unique iPLA1-like protein, Yor022c, in the budding yeast Saccharomyces cerevisiae. By the mass spectrometry analysis, we demonstrate that Yor022c is actually a phospholipase displaying sn-1-specific activity toward phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid, generating 2-acyl lysophospholipids. GFP-fused Yor022c co-stained with the mitochondrial dye MitoTracker, indicating that, unlike its mammalian counterparts, it is a mitochondrial protein. Further biochemical fractionation experiment combined with protease sensitivity assay showed that Yor022c localizes to the mitochondrial matrix. Thus Yor022c is the first PLA1 putatively involved in the maintenance of sn-1 acyl chains of phospholipids in the mitochondrial inner membrane.


Assuntos
Fosfolipases A1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ativação Enzimática , Mitocôndrias , Saccharomyces cerevisiae/ultraestrutura , Distribuição Tecidual
14.
Fungal Genet Biol ; 96: 1-11, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27634187

RESUMO

In mammals, cytosolic phospholipases A2 (cPLA2s) play important physiological roles by releasing arachidonic acid, a precursor for bioactive lipid mediators, from the biological membranes. In contrast, fungal cPLA2-like proteins are much less characterized and their roles have remained elusive. AoPlaA is a cPLA2-like protein in the filamentous fungus Aspergillus oryzae which, unlike mammalian cPLA2, localizes to mitochondria. In this study, we investigated the biochemical and physiological functions of AoPlaA. Recombinant AoPlaA produced in E. coli displayed Ca2+-independent lipolytic activity. Mass spectrometry analysis demonstrated that AoPlaA displayed PLA2 activity to phosphatidylethanolamine (PE), but not to other phospholipids, and generated 1-acylated lysoPE. Catalytic site mutants of AoPlaA displayed almost no or largely reduced activity to PE. Consistent with PE-specific activity of AoPlaA, AoplaA-overexpressing strain showed decreased PE content in the mitochondrial fraction. In contrast, AoplaA-disruption strain displayed increased content of cardiolipin. AoplaA-overexpressing strain, but not its counterparts overexpressing the catalytic site mutants, exhibited retarded growth at low temperature, possibly because of the impairment of the mitochondrial function caused by excess degradation of PE. These results suggest that AoPlaA is a novel PE-specific PLA2 that plays a regulatory role in the maintenance of mitochondrial phospholipid composition.


Assuntos
Aspergillus oryzae/enzimologia , Mitocôndrias/enzimologia , Fosfatidiletanolaminas/metabolismo , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Aspergillus oryzae/crescimento & desenvolvimento , Escherichia coli/genética , Fosfolipases A2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura
15.
J Gen Appl Microbiol ; 62(5): 217-224, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27600355

RESUMO

The complete hydrolysis of lignocellulose requires the actions of a variety of enzymes, including those that cleave the linkage between lignin and hemicellulose. The enzyme glucuronoyl esterase (GE) that constitutes a novel family of carbohydrate esterases, CE15, has been shown to display a unique ability to cleave the ester linkage between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid of hemicellulose. We herein report identification, expression, and functional characterization of a new GE, NcGE, from the filamentous fungus Neurospora crassa. C-terminally c-myc and hexahistidine-tagged NcGE was heterologously expressed in the methylotrophic yeast Pichia pastoris. NcGE purified from the culture supernatant through Ni-NTA and anion exchange chromatographies showed the ability to hydrolyze the substrate 3-(4-methoxyphenyl) propyl methyl 4-O-methyl-α-D-glucopyranosiduronate, which mimics the ester linkage of 4-O-methyl-D-glucuronic acid in lignin-carbohydrate complexes (LCCs). This esterase showed the characteristic of a mesophilic enzyme with the temperature optimum at 40-50°C, and displayed the optimal activity at pH 7 and broad pH stability. Based on the alignment of NcGE with other GEs so far characterized, we propose novel consensus sequences for GEs containing the catalytic triad.


Assuntos
Domínio Catalítico , Sequência Consenso , Esterases/química , Esterases/metabolismo , Ácido Glucurônico/metabolismo , Neurospora crassa/enzimologia , Oligopeptídeos/química , Sequência de Aminoácidos , Catálise , Esterases/genética , Esterases/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hidrólise , Lignina/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Oligopeptídeos/metabolismo , Pichia/genética , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
16.
Protein Expr Purif ; 115: 69-75, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26272369

RESUMO

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca(2+)-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2.


Assuntos
Neurospora crassa/enzimologia , Fosfolipases A2 Secretórias/isolamento & purificação , Fosfolipases A2 Secretórias/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Micelas , Dados de Sequência Molecular , Neurospora crassa/genética , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipases A2 Secretórias/química , Fosfolipases A2 Secretórias/genética , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência
17.
Biochem Biophys Res Commun ; 452(3): 520-5, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25173929

RESUMO

Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.


Assuntos
Mananas/química , Oligossacarídeos/química , Parabasalídeos/química , Polissacarídeos/química , Proteínas de Protozoários/química , beta-Manosidase/química , Animais , Expressão Gênica , Isópteros/fisiologia , Cinética , Mananas/metabolismo , Oligossacarídeos/metabolismo , Parabasalídeos/enzimologia , Pichia/genética , Pichia/metabolismo , Polissacarídeos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Simbiose , beta-Manosidase/genética , beta-Manosidase/metabolismo
18.
J Biol Chem ; 289(15): 10843-10852, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24570006

RESUMO

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of ß-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites -5 and -2, respectively, in addition to accommodation of both glucose and mannose at subsites -3 and -4. RsMan26C has a long open cleft with a hydrophobic platform of Trp(94) at subsite -5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met(85) specifically interacts with the equatorial O-2 of glucose at subsite -3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.


Assuntos
Isópteros/microbiologia , Mananas/metabolismo , Manosidases/metabolismo , Simbiose , beta-Manosidase/metabolismo , Animais , Biomassa , Catálise , Domínio Catalítico , Cromatografia em Camada Fina , Eucariotos/enzimologia , Glucose/metabolismo , Hidrólise , Intestinos/microbiologia , Lignina/metabolismo , Manose/metabolismo , Polissacarídeos/metabolismo , Conformação Proteica , Especificidade por Substrato
19.
Biosci Biotechnol Biochem ; 77(12): 2510-3, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24317073

RESUMO

We found that brain-derived neurotrophic factor (BDNF)-induced phosphorylation of mitogen-activated protein kinase (MAPK) and Akt in cerebellar granule neurons was specifically potentiated by LPC. LPC also augmented the BDNF-induced phosphorylation of TrkB, the receptor for BDNF. In TrkB-transfected CHO-K1 cells, LPC potentiated BDNF-induced MAPK phosphorylation. These results suggest that LPC plays a role in BDNF-TrkB signaling by regulating the activation of TrkB.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Cerebelo/citologia , Lisofosfatidilcolinas/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Receptor trkB/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Camundongos , Fosforilação/efeitos dos fármacos
20.
FEBS Open Bio ; 3: 243-51, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23772401

RESUMO

Lysophosphatidylcholine (LPC) is one of the major lysophospholipids mainly generated by phospholipase A2 (PLA2)-mediated hydrolysis of phosphatidylcholine (PC). We previously found that LPC displays neurotrophin-like activity in the rat pheochromocytoma PC12 cells and in cerebellar granule neurons, but the molecular mechanism remains unclear. We report here that LPC specifically enhances nerve growth factor (NGF)-induced signals in PC12 cells. When PC12 cells were treated with NGF, MAPK was phosphorylated, but this phosphorylation was significantly elevated when LPC was added together. In accordance, NGF-induced expression of immediate early genes, c-fos and NGF-IA, was upregulated by LPC. Phosphorylation of the upstream components, MEK and NGF receptor TrkA, was also promoted by LPC, which was in line with increased phosphorylation of Akt. In contrast, LPC did not enhance epidermal growth factor (EGF)-, basic fibroblast growth factor-, or insulin-like growth factor-1-induced signals. Studies using TrkA/EGF receptor chimeras demonstrated that the extracellular domain, but not the transmembrane or intracellular domains, of TrkA is responsible for the effect of LPC. Exogenously-added secretory PLA2 (sPLA2) enhanced NGF-induced MAPK phosphorylation at a comparable level to LPC, suggesting that LPC generated in situ by sPLA2-mediated hydrolysis of membrane PC stimulated NGF-TrkA signal. Taken together, these results indicate a specific role and function of LPC on NGF-TrkA signaling pathway.

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