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1.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807844

RESUMO

We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from Amycolatopsis sp., Thermomonospora curvata, and Auricularia auricula-judae, VP from Pleurotus eryngii, and LiP from Phanerochaete chrysosporium were produced, and their kinetic constants and reduction potentials determined. Sharp differences were found in the oxidation of nonphenolic simple (veratryl alcohol, VA) and dimeric (veratrylglycerol-ß- guaiacyl ether, VGE) lignin model compounds, with LiP showing the highest catalytic efficiencies (around 15 and 200 s-1·mM-1 for VGE and VA, respectively), while the efficiency of the A. auricula-judae DyP was 1-3 orders of magnitude lower, and no activity was detected with the bacterial DyPs. VP and LiP also showed the highest reduction potential (1.28-1.33 V) in the rate-limiting step of the catalytic cycle (i.e., compound-II reduction to resting enzyme), estimated by stopped-flow measurements at the equilibrium, while the T. curvata DyP showed the lowest value (1.23 V). We conclude that, when using realistic enzyme doses, only fungal LiP and VP, and in much lower extent fungal DyP, oxidize nonphenolic aromatics and, therefore, have the capability to act on the main moiety of the native lignin macromolecule.


Assuntos
Catalase/química , Corantes/química , Proteínas Fúngicas/química , Fungos/enzimologia , Lignina/química , Peroxidase/química
2.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919261

RESUMO

Carbon dioxide (CO2), a vital molecule of the carbon cycle, is a critical component in living organisms' metabolism, performing functions that lead to the building of compounds fundamental for the life cycle. In all living organisms, the CO2/bicarbonate (HCO3-) balancing is governed by a superfamily of enzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the pivotal physiological reaction, consisting of the reversible hydration of the CO2 to HCO3- and protons. Opportunistic and pathogenic fungi can sense the environmental CO2 levels, which influence their virulence or environmental subsistence traits. The fungal CO2-sensing is directly stimulated by HCO3- produced in a CA-dependent manner, which directly activates adenylyl cyclase (AC) involved in the fungal spore formation. The interference with CA activity may impair fungal growth and virulence, making this approach interesting for designing antifungal drugs with a novel mechanism of action: the inhibition of CAs linked to the CO2/HCO3-/pH chemosensing and signaling. This review reports that sulfonamides and their bioisosteres as well as inorganic anions can inhibit in vitro the ß- and α-CAs from the fungi, suggesting how CAs may be considered as a novel "pathogen protein" target of many opportunistic, pathogenic fungi.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/efeitos dos fármacos , Fungos/enzimologia , Sulfonamidas/farmacologia , Antifúngicos/farmacologia , Dióxido de Carbono/metabolismo , Fungos/efeitos dos fármacos
3.
ACS Appl Mater Interfaces ; 13(4): 5111-5124, 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472360

RESUMO

Artificial enzymes with modulated enzyme-mimicking activities of natural systems represent a challenge in catalytic applications. Here, we show the creation of artificial Cu metalloenzymes based on the generation of Cu nanoparticles in an enzyme matrix. Different enzymes were used, and the structural differences between the enzymes especially influenced the controlled the size of the nanoparticles and the environment that surrounds them. Herein, we demonstrated that the oxidase-like catalytic activity of these copper nanozymes was rationally modulated by enzyme used as a scaffold, with a special role in the nanoparticle size and their environment. In this sense, these nanocopper hybrids have confirmed the ability to mimic a unique enzymatic activity completely different from the natural activity of the enzyme used as a scaffold, such as tyrosinase-like activity or as Fenton catalyst, which has extremely higher stability than natural mushroom tyrosinase. More interestingly, the oxidoreductase-like activity of nanocopper hybrids was cooperatively modulated with the synergistic effect between the enzyme and the nanoparticles improving the catalase activity (no peroxidase activity). Additionally, a novel dual (metallic and enzymatic activity) of the nanozyme made the highly improved catechol-like activity interesting for the design of 3,4-dihydroxy-l-phenylalanine (l-DOPA) biosensor for detection of tyrosinase. These hybrids also showed cytotoxic activity against different tumor cells, interesting in biocatalytic tumor therapy.


Assuntos
Materiais Biomiméticos/uso terapêutico , Técnicas Biossensoriais , Cobre/uso terapêutico , Nanopartículas/uso terapêutico , Neoplasias/terapia , Bactérias/enzimologia , Biocatálise , Materiais Biomiméticos/química , Técnicas Biossensoriais/métodos , Cobre/química , Terapia Enzimática/métodos , Fungos/enzimologia , Humanos , Modelos Moleculares , Monofenol Mono-Oxigenase/análise , Nanopartículas/química , Oxirredutases/química , Oxirredutases/uso terapêutico , Conformação Proteica
4.
J Med Chem ; 64(2): 1116-1126, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33356256

RESUMO

Due to the evolution and development of antifungal drug resistance, limited efficacy of existing drugs has led to high mortality in patients with serious fungal infections. To develop novel antifungal therapeutic strategies, herein a series of carboline fungal histone deacetylase (HDAC) inhibitors were designed and synthesized, which had potent synergistic effects with fluconazole against resistant Candida albicans infection. In particular, compound D12 showed excellent in vitro and in vivo synergistic antifungal efficacy with fluconazole to treat azole-resistant candidiasis. It cooperated with fluconazole in reducing the virulence of C. albicans by blocking morphological mutual transformation and inhibiting biofilm formation. Mechanism studies revealed that the reversion of drug resistance was due to downregulation of the expression of the azole target gene ERG11 and efflux gene CDR1. Taken together, fungal HDAC inhibitor D12 offered a promising lead compound for combinational treatment of azole-resistant candidiasis.


Assuntos
Azóis/uso terapêutico , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Carbolinas/síntese química , Carbolinas/uso terapêutico , Farmacorresistência Fúngica/efeitos dos fármacos , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/uso terapêutico , Animais , Biofilmes/efeitos dos fármacos , Candida albicans/enzimologia , Candidíase/microbiologia , Carbolinas/toxicidade , Quimioterapia Combinada , Feminino , Fluconazol/farmacologia , Proteínas Fúngicas/efeitos dos fármacos , Fungos/efeitos dos fármacos , Fungos/enzimologia , Inibidores de Histona Desacetilases/toxicidade , Humanos , Fígado/patologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana
5.
Nutrients ; 12(12)2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33327648

RESUMO

In this investigation, we reported the production of prototype breads from the processed flours of three specific Triticum turgidum wheat genotypes that were selected in our previous investigation for their potential low toxic/immunogenic activity for celiac disease (CD) patients. The flours were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains, and in presence of fungal endoproteases. The breads were characterized by R5 competitive enzyme linked immunosorbent assay in order to quantify the residual gluten, and the differential efficacy in gluten degradation was assessed. In particular, two of them were classified as gluten-free (<20 ppm) and very low-gluten content (<100 ppm) breads, respectively, whereas the third monovarietal prototype retained a gluten content that was well above the safety threshold prescribed for direct consumption by CD patients. In order to investigate such a genotype-dependent efficiency of the detoxification method applied, an advanced proteomic characterization by high-resolution tandem mass spectrometry was performed. Notably, to the best of our knowledge, this is the first proteomic investigation which benefitted, for protein identification, from the full sequencing of the Triticum turgidum ssp. durum genome. The differences of the proteins' primary structures affecting their susceptibility to hydrolysis were investigated. As a confirmation of the previous immunoassay-based results, two out of the three breads made with the processed flours presented an exhaustive degradation of the epitopic sequences that are relevant for CD immune stimulatory activity. The list of the detected epitopes was analyzed and critically discussed in light of their susceptibility to the detoxification strategy applied. Finally, in-vitro experiments of human gastroduodenal digestion were carried out in order to assess, in-silico, the toxicity risk of the prototype breads under investigation for direct consumption by CD patients. This approach allowed us to confirm the total degradation of the epitopic sequences upon gastro-duodenal digestion.


Assuntos
Pão/análise , Farinha/análise , Glutens/análise , Inativação Metabólica , Triticum/química , Doença Celíaca/dietoterapia , Doença Celíaca/metabolismo , Dieta Livre de Glúten/métodos , Digestão , Duodeno , Epitopos , Fermentação , Fungos/enzimologia , Genótipo , Humanos , Hidrólise , Lactobacillus/metabolismo , Proteólise , Proteômica , Estômago
6.
Biochemistry ; 59(51): 4787-4792, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33332106

RESUMO

Biosynthesis of fungal nonribosomal peptides frequently involves redox enzymes such as flavin-containing monooxygenase (FMO) to introduce complexity into the core chemical structure. One such example is the formation of spiro-carbons catalyzed by various oxidases. Because many chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, understanding the mechanism of spiro-carbon biosynthesis is of great interest. We previously identified FqzB, an FMO from the fumiquinazoline biosynthetic pathway responsible for epoxidation of fumiquinazoline F that crosstalks with the fumitremorgin biosynthetic pathway to form spirotryprostatin A via epoxidation of the precursor fumitremorgin C. What makes FqzB more interesting is its relaxed substrate specificity, where it can accept a range of other substrates, including tryprostatins A and B along with its original substrate fumiquinazoline F. Here, we characterized FqzB crystallographically and examined FqzB and its site-specific mutants kinetically to understand how this promiscuous epoxidase works. Furthermore, the mutagenesis studies as well as computational docking experiments between the FqzB crystal structure and its known substrates spirotryprostatin A and B, as well as fumitremorgin C and fumiquinazoline F, provided insight into potential modes of substrate recognition and the source of broad substrate tolerance exhibited by this epoxidase. This study serves as a foundation for further characterization and engineering of this redox enzyme, which has potential utility as a valuable catalyst with broad substrate tolerance and an ability to introduce chemical complexity into carbon frameworks for chemoenzymatic and biosynthetic applications.


Assuntos
Produtos Biológicos/química , Compostos de Epóxi/química , Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Compostos de Espiro/química , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Fungos/enzimologia , Cinética , Oxigenases de Função Mista/genética , Modelos Químicos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Especificidade por Substrato
7.
Ann Agric Environ Med ; 27(4): 562-567, 2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33356061

RESUMO

INTRODUCTION: In recent years, the number of diseases caused by fungal pathogens has increased significantly. Many species of fungi are pathogenic for plants, causing a threat to food production and to humans, and are among the causes of chronic diseases. OBJECTIVE: The aim of the study is to determine the enzyme profiles of fungi, depending on the different types of fruit with which they have contact, and to determine the differences in these profiles in relation to the substrate on which they are grown. MATERIAL AND METHODS: Six strains of fungi identified as Cladosporium sphaerospermum, Fusarium poae, Alternaria alternata, Penicillium expansum, Penicillium verucosum and Acremonium strictum, isolated from fruits, were selected and analyzed for enzymatic profiles. The enzymatic activity was assessed using the API ZYM test (bioMerieux, France). RESULTS: In the majority of the 6 fungal strains isolated from fruits, enzymes belonging to glycol-hydrolases were the most active. The exception was Acremonium strictum, where phosphatases dominated. Among most fungal isolates, the enzymes ß- glucosidase and N-acetyl-ß-glucosaminidase showed the highest activity. The highest ß-glucosidase activities were found in Cladosporium sphaerospermum and Penicillium expansum. On the other hand, lipase, α-fucosidase and α-chymotrypsin showed the least activity. The least activity of these enzymes or their complete absence was observed in Fusarium poae, Alternaria alternata, Penicillium expansum and Acremonium strictum. CONCLUSIONS: The activity of hydrolytic enzymes in the isolated fungi depended on the addition of fruit and the type of medium. Individual fruits can increase or decrease the activity of the enzymes. Fungi present in fruit have pathogenic properties and can be possible risk factors for fungal infections.


Assuntos
Microbiologia de Alimentos , Frutas/química , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Frutas/microbiologia , Proteínas Fúngicas/classificação
8.
Nat Commun ; 11(1): 5786, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188177

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic properties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, with kcat/KmH2O2 values in the order of 105-106 M-1 s-1. Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases.


Assuntos
Celulose/metabolismo , Oxigenases de Função Mista/metabolismo , Bactérias/enzimologia , Catalase/metabolismo , Quitina/metabolismo , Fungos/enzimologia , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/metabolismo , Cinética , Nanopartículas/química , Especificidade por Substrato
9.
Nat Commun ; 11(1): 4864, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978392

RESUMO

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal ß-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.


Assuntos
Tolerância a Medicamentos/fisiologia , Fungos/enzimologia , Fungos/metabolismo , Xilosidases/química , Xilosidases/metabolismo , Biocatálise , Domínio Catalítico , Fungos/efeitos dos fármacos , Glicoconjugados/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese , Especificidade por Substrato , Talaromyces/enzimologia , Talaromyces/genética , Xilosidases/genética
10.
Arch Microbiol ; 202(10): 2809-2824, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32747999

RESUMO

The impact of agricultural land-use on soil microbial community composition and enzyme activity has not been extensively investigated in Ultisols. We investigated soil health parameters by analyzing phospholipid fatty acids (PLFAs), extracellular enzyme activity, C and N stocks, and soil structure. Four land uses were established in a tropical climate region of Brazil: native Cerrado (savanna), monoculture pasture [Urochloa brizantha (Hochst. Ex A. Rich.) R. Webster 'Marandu'], an integrated crop-livestock system (ICLS), and maize (Zea mays)-fallow in a no-tillage system. Soil microbial biomass was 40% higher in the native Cerrado than in the monoculture pasture, ICLS, and no-tillage maize. Soil organic carbon was positively correlated with microbial community composition (MB; gram-; AC; AMF; Fungi; F: B ratio) and enzyme activity (bG, AP, NAG). Large macroaggregates were positively correlated with bG, AP, and AMF. In summary, the native Cerrado had a higher level of carbon at the soil surface and greater soil structure with increased microbial biomass, gram+ bacteria, AMF, fungi, and F:B ratio in a tropical region of Brazil. However, bG and AP enzyme activities were lower in the ICLS and no-till maize at the soil surface (0-5 cm) compared to the native Cerrado. The conversion of native Cerrado to agricultural systems shifted the soil microbial community composition, enzyme activity, C and N, and soil structure of this sandy soil of the Brazilian Cerrado.


Assuntos
Bactérias/isolamento & purificação , Fungos/isolamento & purificação , Microbiota/fisiologia , Microbiologia do Solo , Solo/química , Agricultura , Bactérias/enzimologia , Biomassa , Brasil , Carbono/análise , Ácidos Graxos/análise , Fungos/enzimologia , Nitrogênio/análise , Clima Tropical , Zea mays/microbiologia
11.
Food Chem ; 332: 127417, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32629332

RESUMO

The use of UHPH sterilization in the absence of SO2 has been used to eliminate wild microorganisms and inactivate oxidative enzymes. A white must of the Muscat of Alexandria grape variety was continuously processed by UHPH at 300 MPa (inlet temperature: 23-25 °C). The initial microbial load of the settled must was 4-log CFU/mL for both yeast and moulds, and slightly lower for bacteria. After UHPH processing, no microorganisms were detected in 1 mL. UHPH musts remain without fermentative activity for more than 60 days. Concentrations of the thermal markers indicated the absence of thermal damage in the UHPH-treated musts, since 5-hydroxymethylfurfural was not detected. In addition, the must treated by UHPH keeps terpene concentrations similar to those of the untreated controls. A strong inactivation of the oxidative enzymes was observed, with no browning at room temperature for more than 3 days. The antioxidant value of the UHPH-treated must was 156% higher than the control.


Assuntos
Bactérias/crescimento & desenvolvimento , Microbiologia de Alimentos/métodos , Fungos/crescimento & desenvolvimento , Esterilização/métodos , Vinho/análise , Bactérias/enzimologia , Bactérias/metabolismo , Fermentação , Manipulação de Alimentos , Fungos/enzimologia , Fungos/genética , Fungos/metabolismo , Humanos , Dióxido de Enxofre/análise , Paladar , Temperatura , Vitis/química
12.
Proc Natl Acad Sci U S A ; 117(21): 11551-11558, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404424

RESUMO

As the primary decomposers of organic material in terrestrial ecosystems, fungi are critical agents of the global carbon cycle. Yet our ability to link fungal community composition to ecosystem functioning is constrained by a limited understanding of the factors accounting for different wood decomposition rates among fungi. Here we examine which traits best explain fungal decomposition ability by combining detailed trait-based assays on 34 saprotrophic fungi from across North America in the laboratory with a 5-y field study comprising 1,582 fungi isolated from 74 decomposing logs. Fungal growth rate (hyphal extension rate) was the strongest single predictor of fungal-mediated wood decomposition rate under laboratory conditions, and accounted for up to 27% of the in situ variation in decomposition in the field. At the individual level, decomposition rate was negatively correlated with moisture niche width (an indicator of drought stress tolerance) and with the production of nutrient-mineralizing extracellular enzymes. Together, these results suggest that decomposition rates strongly align with a dominance-tolerance life-history trade-off that was previously identified in these isolates, forming a spectrum from slow-growing, stress-tolerant fungi that are poor decomposers to fast-growing, highly competitive fungi with fast decomposition rates. Our study illustrates how an understanding of fungal trait variation could improve our predictive ability of the early and midstages of wood decay, to which our findings are most applicable. By mapping our results onto the biogeographic distribution of the dominance-tolerance trade-off across North America, we approximate broad-scale patterns in intrinsic fungal-mediated wood decomposition rates.


Assuntos
Fungos/fisiologia , Madeira/microbiologia , Ciclo do Carbono/fisiologia , Ecossistema , Fungos/classificação , Fungos/enzimologia , Hifas/fisiologia , Micobioma/fisiologia , América do Norte
13.
Appl Environ Microbiol ; 86(14)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414792

RESUMO

Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ∼500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidation, a ß-diketone initiator commonly used in vinyl polymerization reactions.IMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond doubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), and protein engineering (in particular, directed evolution) has become the key driver for adaptation of these enzymes to harsh industrial conditions. Usually, the first requirement for directed laccase evolution is heterologous expression, which presents an important hurdle and often a time-consuming process. In this work, we resurrected a fungal Mesozoic laccase node which showed strikingly high heterologous expression and pH stability. As a proof of concept that the ancestral laccase is a suitable blueprint for engineering, we performed a quick directed evolution campaign geared to the oxidation of the ß-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers.


Assuntos
Evolução Molecular , Fungos/genética , Lacase/genética , Fungos/enzimologia , Micologia , Paleontologia
14.
J Nat Med ; 74(3): 501-512, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32180104

RESUMO

Aromatic prenyltransferases (PTases), including ABBA-type and dimethylallyl tryptophan synthase (DMATS)-type enzymes from bacteria and fungi, play important role for diversification of the natural products and improvement of the biological activities. For a decade, the characterization of enzymes and enzymatic synthesis of prenylated compounds by using ABBA-type and DMATS-type PTases have been demonstrated. Here, I introduce several examples of the studies on chemoenzymatic synthesis of unnatural prenylated compounds and the enzyme engineering of ABBA-type and DMATS-type PTases.


Assuntos
Alquil e Aril Transferases/metabolismo , Bactérias/enzimologia , Dimetilaliltranstransferase/metabolismo , Fungos/enzimologia , Engenharia de Proteínas , Bactérias/metabolismo , Produtos Biológicos/metabolismo , Fungos/metabolismo , Prenilação/fisiologia
15.
World J Microbiol Biotechnol ; 36(4): 53, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32172335

RESUMO

The recent scientific progresses on the use of enzyme-mediated reactions in organic, non-aqueous and aqueous media have significantly supported the growing demand of new biotechnological and/or pharmacological products. Today, a plethora of microbial enzymes, used as biocatalysts, are available. Among these, microbial transglutaminases (MTGs) are broadly used for their ability to catalyse the formation of an isopeptide bond between the γ-amide group of glutamines and the ε-amino group of lysine. Due to their promiscuity towards primary amine-containing substrates and the more stringent specificity for glutamine-containing peptide sequences, several combined approaches can be tailored for different settings, making MTGs very attractive catalysts for generating protein-protein and protein small molecule's conjugates. The present review offers a recent update on the modifications attainable by MTG-catalysed bioreactions as reported between 2014 and 2019. In particular, we present a detailed and comparative overview on the MTG-based methods for proteins and antibodies engineering, with a particular outlook on the synthesis of homogeneous antibody-drug conjugates.


Assuntos
Bactérias/enzimologia , Fungos/enzimologia , Engenharia de Proteínas/métodos , Transglutaminases/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Proteínas Fúngicas/metabolismo , Imunoconjugados/metabolismo , Especificidade por Substrato
16.
Biochemistry ; 59(11): 1163-1172, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32135062

RESUMO

Arbuscular mycorrhiza (AM) fungi deliver mineral nutrients to the plant host in exchange for reduced carbon in the form of sugars and lipids. Colonization with AM fungi upregulates a specific host lipid synthesis pathway resulting in the production of fatty acids. Predominantly palmitic acid (16:0) and the unusual palmitvaccenic acid (16:1Δ11cis) accumulate in the fungus Rhizophagus irregularis. Here, we present the isolation and characterization of RiOLE1-LIKE, the desaturase involved in palmitvaccenic acid synthesis, by heterologous expression in yeast and plants. Results are in line with the scenario in which RiOLE1-LIKE encodes an acyl-CoA desaturase with substrate specificity for C15-C18 acyl groups, in particular C16. Phylogenetic analysis of RiOLE1-LIKE-related sequences revealed that this gene is conserved in AM fungi from the Glomales and Diversisporales but is absent from nonsymbiotic Mortierellaceae and Mucoromycotina fungi, suggesting that 16:1Δ11cis provides a specific function during AM colonization.


Assuntos
Ácidos Graxos Dessaturases/metabolismo , Proteínas Fúngicas/metabolismo , Glomeromycota/enzimologia , Micorrizas/enzimologia , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Glomeromycota/química , Glomeromycota/genética , Glomeromycota/metabolismo , Micorrizas/química , Micorrizas/genética , Micorrizas/metabolismo , Ácidos Palmíticos/química , Ácidos Palmíticos/metabolismo , Filogenia
17.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32024019

RESUMO

Discovered in 1883, laccase is one of the first enzymes ever described. Now, after almost 140 years of research, it seems that this copper-containing protein with a number of unique catalytic properties is widely distributed across all kingdoms of life. Laccase belongs to the superfamily of multicopper oxidases (MCOs)-a group of enzymes comprising many proteins with different substrate specificities and diverse biological functions. The presence of cupredoxin-like domains allows all MCOs to reduce oxygen to water without producing harmful byproducts. This review describes structural characteristics and plausible evolution of laccase in different taxonomic groups. The remarkable catalytic abilities and broad substrate specificity of laccases are described in relation to other copper-containing MCOs. Through an exhaustive analysis of laccase roles in different taxa, we find that this enzyme evolved to serve an important, common, and protective function in living systems.


Assuntos
Lacase/química , Lacase/metabolismo , Bactérias/enzimologia , Bactérias/genética , Evolução Molecular , Fungos/enzimologia , Fungos/genética , Humanos , Lacase/genética , Filogenia , Domínios Proteicos , Especificidade por Substrato
18.
Anal Chim Acta ; 1101: 9-22, 2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32029123

RESUMO

With a substantial demand for new anti-obesity drugs for the treatment of obesity, screening lipase inhibitors from natural products has become a popular approach toward drug discovery. Due to the significant advantages of excellent reusability, stability and endurance in extreme pH and temperature conditions, lipase immobilization has been employed as a promising strategy to screen lipase inhibitors. Support is a key factor in the process of enzyme immobilization used to provide excellent biocompatibility, stable physical and chemical properties and abundant binding sites for enzymes. Thus, various supports, including nanofibers, polymeric monoliths, mesoporous materials, nanomaterials, membrane and cellulose paper, are systematically introduced and discussed in this review. Considering these supports, the application of the immobilization of lipase in screening compounds from natural products is also comprehensively reviewed, and the outlook for future research directions is described.


Assuntos
Fármacos Antiobesidade/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Enzimas Imobilizadas/química , Lipase/química , Animais , Fármacos Antiobesidade/química , Biocatálise , Burkholderia cepacia/enzimologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Enzimas Imobilizadas/antagonistas & inibidores , Fungos/enzimologia , Lipase/antagonistas & inibidores , Estruturas Metalorgânicas/química , Nanoestruturas/química , Plantas/química
19.
Arch Microbiol ; 202(5): 967-981, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32052094

RESUMO

L-asparaginase (E.C.3.5.1.1) is an important enzyme that has been purified and characterized for over decades to study and evaluate its anti-carcinogenic activity against different lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma. The ability of the enzyme to convert L-asparagine into aspartic acid and ammonia is the reason behind its anti-cancerous activity. Apart from its medicinal uses, it is widely used in food industry to tackle acrylamide, a probable human carcinogen and, production in carbohydrate-rich foods cooked at high temperatures. There are variety of organisms including microorganisms such as bacteria, fungi, algae, and plants that produce L-asparaginase. The enzyme obtained from different microbial and plant sources have different physiochemical properties and kinetic parameters. L-asparaginases have an optimum pH range between 6 and 10 and an optimum temperature between 37 and 85 °C. This article has reviewed the lowest molecular mass for L-asparaginase in Yersinia pseudotuberculosis Q66CJ2 which is 36.27 kDa, while the highest for Pseudomonas otitidis which has a molecular mass of 205 ± 3 kDa. This review is an attempt to summarize most of the available sources, their phylogenetic relationships, purification methods, data regarding different physiochemical and kinetic properties of L-asparaginase.


Assuntos
Asparaginase/química , Bactérias/enzimologia , Fungos/enzimologia , Doença de Hodgkin/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Amônia/metabolismo , Asparaginase/genética , Asparaginase/isolamento & purificação , Asparagina/química , Ácido Aspártico/metabolismo , Humanos , Filogenia , Plantas
20.
Sci Rep ; 10(1): 1315, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992763

RESUMO

Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs. Surprisingly, most putative ancestors could be expressed as functional tetramers in Escherichia coli and thus retained their ability to oligomerize. All ancestral enzymes displayed increased thermostability compared to both modern enzymes, however, the increase in thermostability was accompanied by a loss in catalytic turnover. One reconstructed ancestral enzyme in particular could be interesting for further drug development, as its ratio of specific activities is more favourable towards tyrosine and it is more thermostable than both modern enzymes. Moreover, long-term stability assessment showed that this variant retained substantially more activity after prolonged incubation at 25 °C and 37 °C, as well as an increased resistance to incubation at 60 °C. Both of these factors are indicative of an extended shelf-life of biopharmaceuticals. We believe that ancestral sequence reconstruction has potential for enhancing the properties of enzyme therapeutics, especially with respect to stability. This work further illustrates that resurrection of putative ancestral oligomeric proteins is feasible and provides insight into the extent of conservation of a functional oligomerization surface area from ancestor to modern enzyme.


Assuntos
Suplementos Nutricionais , Terapia de Reposição de Enzimas , Fenilalanina Amônia-Liase/uso terapêutico , Tirosinemias/terapia , Animais , Ativação Enzimática , Terapia de Reposição de Enzimas/métodos , Estabilidade Enzimática , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Humanos , Cinética , Modelos Moleculares , Fenilalanina Amônia-Liase/administração & dosagem , Fenilalanina Amônia-Liase/química , Fenilalanina Amônia-Liase/classificação , Conformação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Termodinâmica , Tirosinemias/etiologia
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