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1.
PLoS One ; 17(10): e0275023, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36264993

RESUMO

Klebsiella pneumoniae is a bacterial pathogen that is increasingly responsible for hospital-acquired pneumonia and sepsis. Progressive development of antibiotic resistance has led to higher mortality rates and creates a need for novel treatments. Because of the essential role that nucleotides play in many bacterial processes, enzymes involved in purine and pyrimidine metabolism and transport are ideal targets for the development of novel antibiotics. Herein we describe the structure of K. pneumoniae adenosine monophosphate nucleosidase (KpAmn), a purine salvage enzyme unique to bacteria, as determined by cryoelectron microscopy. The data detail a well conserved fold with a hexameric overall structure and clear density for the putative active site residues. Comparison to the crystal structures of homologous prokaryotic proteins confirms the presence of many of the conserved structural features of this protein yet reveals differences in distal loops in the absence of crystal contacts. This first cryo-EM structure of an Amn enzyme provides a basis for future structure-guided drug development and extends the accuracy of structural characterization of this family of proteins beyond this clinically relevant organism.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Humanos , Microscopia Crioeletrônica , N-Glicosil Hidrolases , Antibacterianos , Purinas , Nucleotídeos , Monofosfato de Adenosina , Pirimidinas , Infecções por Klebsiella/microbiologia
2.
Toxins (Basel) ; 14(9)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36136551

RESUMO

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.


Assuntos
Ricina , Sambucus nigra , Sambucus , Adenina , Sequência de Aminoácidos , Galactose , N-Glicosil Hidrolases/genética , Folhas de Planta/metabolismo , Lectinas de Plantas/farmacologia , Proteínas de Plantas/genética , Plantas/metabolismo , RNA Ribossômico , Proteínas Inativadoras de Ribossomos/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Ribossomos/metabolismo , Ricina/metabolismo , Sambucus nigra/genética , Sambucus nigra/metabolismo
3.
Nat Commun ; 13(1): 5331, 2022 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-36088455

RESUMO

Tropical legumes transport fixed nitrogen in form of ureides (allantoin and allantoate) over long distances from the nodules to the shoot. Ureides are formed in nodules from purine mononucleotides by a partially unknown reaction network that involves bacteroid-infected and uninfected cells. Here, we demonstrate by metabolic analysis of CRISPR mutant nodules of Phaseolus vulgaris defective in either xanthosine monophosphate phosphatase (XMPP), guanosine deaminase (GSDA), the nucleoside hydrolases 1 and 2 (NSH1, NSH2) or xanthine dehydrogenase (XDH) that nodule ureide biosynthesis involves these enzymes and requires xanthosine and guanosine but not inosine monophosphate catabolism. Interestingly, promoter reporter analyses revealed that XMPP, GSDA and XDH are expressed in infected cells, whereas NSH1, NSH2 and the promoters of the downstream enzymes urate oxidase (UOX) and allantoinase (ALN) are active in uninfected cells. The data suggest a complex cellular organization of ureide biosynthesis with three transitions between infected and uninfected cells.


Assuntos
Nitrogênio , Phaseolus , Alantoína/metabolismo , N-Glicosil Hidrolases/metabolismo , Nitrogênio/metabolismo , Phaseolus/genética , Xantina Desidrogenase/metabolismo
4.
Biochemistry ; 61(17): 1853-1861, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-35994320

RESUMO

Trichomonas vaginalis is the causative parasitic protozoan of the disease trichomoniasis, the most prevalent, nonviral sexually transmitted disease in the world. T. vaginalis is a parasite that scavenges nucleosides from the host organism via catalysis by nucleoside hydrolase (NH) enzymes to yield purine and pyrimidine bases. One of the four NH enzymes identified within the genome of T. vaginalis displays unique specificity toward purine nucleosides, adenosine and guanosine, but not inosine, and atypically shares greater sequence similarity to the pyrimidine hydrolases. Bioinformatic analysis of this enzyme, adenosine/guanosine-preferring nucleoside ribohydrolase (AGNH), was incapable of identifying the residues responsible for this uncommon specificity, highlighting the need for structural information. Here, we report the X-ray crystal structures of holo, unliganded AGNH and three additional structures of the enzyme bound to fragment and small-molecule inhibitors. Taken together, these structures facilitated the identification of residue Asp231, which engages in substrate interactions in the absence of those residues that typically support the canonical purine-specific tryptophan-stacking specificity motif. An altered substrate-binding pose is mirrored by repositioning within the protein scaffold of the His80 general acid/base catalyst. The newly defined structure-determined sequence markers allowed the assignment of additional NH orthologs, which are proposed to exhibit the same specificity for adenosine and guanosine alone and further delineate specificity classes for these enzymes.


Assuntos
N-Glicosil Hidrolases , Parasitos , Adenosina/química , Animais , Guanosina , Inosina/metabolismo , N-Glicosil Hidrolases/química , Parasitos/metabolismo , Pirimidinas , Especificidade por Substrato
5.
J Vis Exp ; (185)2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35938815

RESUMO

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.


Assuntos
Coccidiose , Neospora , Robótica , Toxoplasma , Animais , Anticorpos Antiprotozoários , Bovinos , Coccidiose/parasitologia , Coccidiose/veterinária , Cães , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Hidrolases , N-Glicosil Hidrolases , Nucleosídeos , Polifosfatos , Gravidez
6.
Toxins (Basel) ; 14(8)2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-36006228

RESUMO

Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new single-chain (type-1) RIPs named sodins were isolated from the seeds (five proteins), edible leaves (one protein) and roots (one protein) of Salsola soda L. Sodins are able to release Endo's fragment when incubated with rabbit and yeast ribosomes and inhibit protein synthesis in cell-free systems (IC50 = 4.83-79.31 pM). In addition, sodin 5, the major form isolated from seeds, as well as sodin eL and sodin R, isolated from edible leaves and roots, respectively, display polynucleotide:adenosine glycosylase activity and are cytotoxic towards the Hela and COLO 320 cell lines (IC50 = 0.41-1200 nM), inducing apoptosis. The further characterization of sodin 5 reveals that this enzyme shows a secondary structure similar to other type-1 RIPs and a higher melting temperature (Tm = 76.03 ± 0.30 °C) and is non-glycosylated, as other sodins are. Finally, we proved that sodin 5 possesses antifungal activity against Penicillium digitatum.


Assuntos
Salsola , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , N-Glicosil Hidrolases/química , Proteínas de Plantas/química , Coelhos , Proteínas Inativadoras de Ribossomos/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1 , Ribossomos/metabolismo , Salsola/metabolismo
7.
Biochemistry ; 61(17): 1883-1893, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-35969806

RESUMO

Enzyme-catalyzed hydrolysis is a fundamental chemical transformation involved in many essential metabolic processes. The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of adenosine-containing metabolites in cysteine and methionine metabolism. Although MTAN enzymes contain highly similar active site architecture and generally follow a dissociative (DN*AN) reaction mechanism, substantial differences in reaction rates and chemical transition state structures have been reported. To understand how subtle changes in sequence and structure give rise to differences in chemistry between homologous enzymes, we have probed the reaction coordinates of two MTAN enzymes using quantum mechanical/molecular mechanical and molecular dynamics simulations combined with experimental methods. We show that the transition state structure and energy are significantly affected by the recruitment and positioning of the catalytic water molecule and that subtle differences in the noncatalytic active site residues alter the environment of the catalytic water, leading to changes in the reaction coordinate and observed reaction rate.


Assuntos
N-Glicosil Hidrolases , Água , Catálise , Desoxiadenosinas , Hidrólise , N-Glicosil Hidrolases/química , Purina-Núcleosídeo Fosforilase , Tionucleosídeos
8.
Toxins (Basel) ; 14(7)2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35878187

RESUMO

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.


Assuntos
Saponaria , Vaccaria , N-Glicosil Hidrolases/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Isoformas de Proteínas , Proteínas Inativadoras de Ribossomos/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/química , Ribossomos/metabolismo , Saponaria/química , Saponaria/metabolismo , Sementes/química
9.
Curr Protein Pept Sci ; 23(4): 211-225, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35598240

RESUMO

S-adenosylhomocysteine nucleosidase (MTAN) is a protein that plays a crucial role in several pathways of bacteria that are essential for its survival and pathogenesis. In addition to the role of MTAN in methyl-transfer reactions, methionine biosynthesis, and polyamine synthesis, MTAN is also involved in bacterial quorum sensing (QS). In QS, chemical signaling autoinducer (AI) secreted by bacteria assists cell to cell communication and is regulated in a cell density-dependent manner. They play a significant role in the formation of bacterial biofilm. MTAN plays a major role in the synthesis of these autoinducers. Signaling molecules secreted by bacteria, i.e., AI-1 are recognized as acylated homoserine lactones (AHL) that function as signaling molecules within bacteria. QS enables bacteria to establish physical interactions leading to biofilm formation. The formation of biofilm is a primary reason for the development of multidrug-resistant properties in pathogenic bacteria like Enterococcus faecalis (E. faecalis). In this regard, inhibition of E. faecalis MTAN (EfMTAN) will block the QS and alter the bacterial biofilm formation. In addition to this, it will also block methionine biosynthesis and many other critical metabolic processes. It should also be noted that inhibition of EfMTAN will not have any effect on human beings as this enzyme is not present in humans. This review provides a comprehensive overview of the structural-functional relationship of MTAN. We have also highlighted the current status, enigmas that warrant further studies, and the prospects for identifying potential inhibitors of EfMTAN for the treatment of E. faecalis infections. In addition to this, we have also reported structural studies of EfMTAN using homology modeling and highlighted the putative binding sites of the protein.


Assuntos
N-Glicosil Hidrolases , Percepção de Quorum , Bactérias/metabolismo , Biofilmes , Homocisteína , Humanos , Metionina , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo
10.
Metallomics ; 14(6)2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35485745

RESUMO

Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for life. These metalloenzymes rely on a wide range of metallocofactors, from single metal ions to complicated metallic clusters. Incorporation of metal ions and metallocofactors into apo-proteins often requires the assistance of proteins known as metallochaperones. Nucleoside triphosphate hydrolases (NTPases) are one important class of metallochaperones and are found widely distributed throughout the domains of life. These proteins use the binding and hydrolysis of nucleoside triphosphates, either adenosine triphosphate or guanosine triphosphate, to carry out highly specific and regulated roles in the process of metalloenzyme maturation. Here, we review recent literature on NTPase metallochaperones and describe the current mechanistic proposals and available structural data. By using representative examples from each type of NTPase, we also illustrate the challenges in studying these complicated systems. We highlight open questions in the field and suggest future directions. This minireview is part of a special collection of articles in memory of Professor Deborah Zamble, a leader in the field of nickel biochemistry.


Assuntos
Metalochaperonas , Metaloproteínas , Trifosfato de Adenosina/metabolismo , Hidrolases , Metalochaperonas/metabolismo , Metais/metabolismo , N-Glicosil Hidrolases , Nucleosídeo-Trifosfatase , Nucleosídeos , Polifosfatos
11.
Toxins (Basel) ; 14(3)2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-35324681

RESUMO

Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed 'molecular surgery', with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer's Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.


Assuntos
Disciplinas das Ciências Biológicas , Imunotoxinas , Indicadores e Reagentes , Ligantes , N-Glicosil Hidrolases , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas
12.
J Pharm Biomed Anal ; 211: 114614, 2022 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-35123329

RESUMO

In Leishmania donovani, the causative protozoan of visceral leishmaniasis, nucleoside hydrolase enzyme (NH) is fundamental for the biosynthesis of its DNA and RNA. Therefore, LdNH is considered a potential target for the development of new leishmaniasis chemotherapy. Moringa oleifera Lamarck is a medicinal plant native to northeastern India with numerous pharmacological properties, including antileishmanial activity. Thus, this study aimed to explore the inhibitory activity of different extracts from M. oleifera leaves and flowers on LdNH. Using LdNH covalently immobilized on magnetic particles (LdNH-MPs), a novel activity assay was developed based on the direct quantification of the formed product by HPLC-DAD. This study screened 12 extracts from leaves and flowers of M. oleifera using different extraction methods. The hydroethanolic (70% ethanol) extract from flowers, obtained by infusion (FIEH) or ultrasound-assisted extraction (FUEH), exhibited respectively IC50 values of 26.2 ± 4.63 µg/mL and 4.96 ± 0.52 µg/mL. The most promising extract (FUEH) was investigated by high-resolution LdNH inhibition profiling, which showed different regions of inhibition in the biochromatogram. A ligand fishing assay was attempted to pinpoint the bioactive compounds. Experimental conditions employed in the elution step of the ligand fishing assay did not result in ligands isolation. However, the analyses of the crude extract solution and the supernatants after the incubation with the active and inactive LdNH-MPs indicated missing peaks referring to compounds selectively retained in the active LdNH-MPs incubation. The missing peaks eluted in the same region that exhibits inhibition in the high-resolution LdNH inhibition profiling. The ligands were identified by UHPLC-MS/MS as palatinose, adenosine, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, hyperoside, quercetin-3-O-malonyl glycoside, and kaempferol-3-O-galactoside.


Assuntos
Moringa oleifera , Ligantes , N-Glicosil Hidrolases , Extratos Vegetais/análise , Folhas de Planta/química , Espectrometria de Massas em Tandem
13.
Biochem J ; 479(4): 463-477, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35175282

RESUMO

ADP-ribosylation has primarily been known as post-translational modification of proteins. As signalling strategy conserved in all domains of life, it modulates substrate activity, localisation, stability or interactions, thereby regulating a variety of cellular processes and microbial pathogenicity. Yet over the last years, there is increasing evidence of non-canonical forms of ADP-ribosylation that are catalysed by certain members of the ADP-ribosyltransferase family and go beyond traditional protein ADP-ribosylation signalling. New macromolecular targets such as nucleic acids and new ADP-ribose derivatives have been established, notably extending the repertoire of ADP-ribosylation signalling. Based on the physiological relevance known so far, non-canonical ADP-ribosylation deserves its recognition next to the traditional protein ADP-ribosylation modification and which we therefore review in the following.


Assuntos
ADP-Ribosilação/fisiologia , ADP Ribose Transferases/química , ADP Ribose Transferases/classificação , ADP Ribose Transferases/fisiologia , Difosfato de Adenosina/metabolismo , Guanosina/metabolismo , N-Glicosil Hidrolases/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Transdução de Sinais , Relação Estrutura-Atividade , Timidina/metabolismo , Ubiquitina/metabolismo
14.
Mol Cancer ; 21(1): 16, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35031058

RESUMO

BACKGROUND: Gliomas are the most common malignant primary brain tumours with a highly immunosuppressive tumour microenvironment (TME) and poor prognosis. Circular RNAs (circRNA), a newly found type of endogenous noncoding RNA, characterized by high stability, abundance, conservation, have been shown to play an important role in the pathophysiological processes and TME remodelling of various tumours. METHODS: CircRNA sequencing analysis was performed to explore circRNA expression profiles in normal and glioma tissues. The biological function of a novel circRNA, namely, circNEIL3, in glioma development was confirmed both in vitro and in vivo. Mechanistically, RNA pull-down, mass spectrum, RNA immunoprecipitation (RIP), luciferase reporter, and co-immunoprecipitation assays were conducted. RESULTS: We identified circNEIL3, which could be cyclized by EWS RNA-binding protein 1(EWSR1), to be upregulated in glioma tissues and to correlate positively with glioma malignant progression. Functionally, we confirmed that circNEIL3 promotes tumorigenesis and carcinogenic progression of glioma in vitro and in vivo. Mechanistically, circNEIL3 stabilizes IGF2BP3 (insulin-like growth factor 2 mRNA binding protein 3) protein, a known oncogenic protein, by preventing HECTD4-mediated ubiquitination. Moreover, circNEIL3 overexpression glioma cells drives macrophage infiltration into the tumour microenvironment (TME). Finally, circNEIL3 is packaged into exosomes by hnRNPA2B1 and transmitted to infiltrated tumour associated macrophages (TAMs), enabling them to acquire immunosuppressive properties by stabilizing IGF2BP3 and in turn promoting glioma progression. CONCLUSIONS: This work reveals that circNEIL3 plays a nonnegligible multifaceted role in promoting gliomagenesis, malignant progression and macrophage tumour-promoting phenotypes polarization, highlighting that circNEIL3 is a potential prognostic biomarker and therapeutic target in glioma.


Assuntos
Exossomos/metabolismo , Glioma/etiologia , Glioma/metabolismo , Macrófagos/metabolismo , N-Glicosil Hidrolases/genética , RNA Circular/genética , Proteína EWS de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Suscetibilidade a Doenças , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Imunomodulação , Macrófagos/imunologia , Masculino , Camundongos , Modelos Biológicos , N-Glicosil Hidrolases/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteína EWS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Relação Estrutura-Atividade , Ubiquitina/metabolismo
15.
ACS Chem Biol ; 17(1): 17-23, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-34904435

RESUMO

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen that identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib inhibits SARS-CoV-2 and MERS-CoV Mac1 but not the closest human homologue, MacroD2. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for the screening of large compound libraries to identify improved macrodomain inhibitors and to explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.


Assuntos
Antivirais/farmacologia , Ensaios de Triagem em Larga Escala/métodos , N-Glicosil Hidrolases/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Dasatinibe/farmacologia , Domínios Proteicos , SARS-CoV-2/enzimologia
16.
PLoS One ; 16(11): e0259465, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34731210

RESUMO

Curtobacterium sp. GD1 was isolated from leaves of conventionally grown soybean in Brazil. It was noteworthy that among all bacteria previously isolated from the same origin, only Curtobacterium sp. GD1 showed a strong chitinase activity. The enzyme was secreted and its production was induced by the presence of colloidal chitin in the medium. The chitinase was partially purified and characterized: molecular weight was approximately 37 kDa and specific activity 90.8 U/mg. Furthermore, Curtobacterium sp. GD1 genome was sequenced and analyzed. Our isolate formed a phylogenetic cluster with four other Curtobacterium spp. strains, with ANIb/ANIm ≥ 98%, representing a new, still non described Curtobacterium species. The circular genome visualization and comparison of genome sequences of strains forming new cluster indicated that most regions within their genomes were highly conserved. The gene associated with chitinase production was identified and the distribution pattern of glycosyl hydrolases genes was assessed. Also, genes associated with catabolism of structural carbohydrates such as oligosaccharides, mixed polysaccharides, plant and animal polysaccharides, as well as genes or gene clusters associated with resistance to antibiotics, toxic compounds and auxin biosynthesis subsystem products were identified. The abundance of putative glycosyl hydrolases in the genome of Curtobacterium sp. GD1 suggests that it has the tools for the hydrolysis of different polysaccharides. Therefore, Curtobacterium sp. GD1 isolated from soybean might be a bioremediator, biocontrol agent, an elicitor of the plant defense responses or simply degrader.


Assuntos
Actinobacteria/fisiologia , Quitina/química , Quitinases/genética , Soja/microbiologia , Sequenciamento Completo do Genoma/métodos , Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Hidrólise , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Filogenia , Folhas de Planta/química , Folhas de Planta/microbiologia , Soja/metabolismo
17.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34266952

RESUMO

The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Células Germinativas Vegetais/enzimologia , Meristema/enzimologia , N-Glicosil Hidrolases/metabolismo , Transativadores/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Diferenciação Celular , Proliferação de Células , Células Germinativas Vegetais/citologia , Meristema/genética , Meristema/crescimento & desenvolvimento , N-Glicosil Hidrolases/genética , Transativadores/genética
18.
Sci Rep ; 11(1): 11803, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083602

RESUMO

Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.


Assuntos
Enzimas/química , Lignina/química , Espectrometria de Massas/métodos , N-Glicosil Hidrolases/química , Ativação Enzimática , Ensaios Enzimáticos , Estrutura Molecular
19.
Biochemistry ; 60(24): 1933-1946, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34077175

RESUMO

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.


Assuntos
Helicobacter pylori/metabolismo , Nucleosídeos/metabolismo , Vitamina K 2/metabolismo , Domínio Catalítico , Cristalografia por Raios X/métodos , Desoxiadenosinas , Helicobacter pylori/química , Helicobacter pylori/enzimologia , Modelos Moleculares , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Nucleosídeos/química , Purina-Núcleosídeo Fosforilase/química , Especificidade por Substrato , Tionucleosídeos , Vitamina K 2/análogos & derivados
20.
Nat Commun ; 12(1): 2739, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016974

RESUMO

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.


Assuntos
Arabidopsis/imunologia , Cloroplastos/imunologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Epiderme Vegetal/imunologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Auxilinas/genética , Auxilinas/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Colletotrichum/imunologia , Colletotrichum/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Magnaporthe/imunologia , Magnaporthe/patogenicidade , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Epiderme Vegetal/microbiologia , Folhas de Planta/citologia , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/imunologia , Pseudomonas syringae/patogenicidade
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