RESUMO
Cyanase is a possible solution to reduce the environmental impact of cyanide. However, the enzyme's dependence on HCO3- limits its industrial applications. To overcome this problem, carbonic anhydrase is utilized in this study. Three types of Catcher/Tag systems were introduced into the cyanase (psCYN) from Pseudomonas stutzeri and the carbonic anhydrase (hmCA) from Hydrogenovibrio marinus to construct enzyme complexes via irreversible covalent bonds. Initially, a cyanase complex with the aid of scaffolding proteins was designed. The results of cyanase complexes using scaffolding proteins were similar to or inferior to those of the two free enzymes. To address this, the two enzymes were manipulated to form a direct bioconjugation without the need for scaffolding proteins. The two enzymes forming a direct conjugation showed activity more than 2.5 times higher than that of cyanase alone. In conclusion, this outcome will contribute to solving problems related to residual cyanides in food and the environment.
Assuntos
Anidrases Carbônicas , Cianetos/metabolismo , Cianatos/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Complexos MultienzimáticosRESUMO
SUMOylation is a post-translational modification of proteins that regulates these proteins' localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Mutação , Sumoilação/fisiologia , Animais , Biomarcadores Tumorais , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Cromatina , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Instabilidade Genômica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Sumoilação/efeitos dos fármacos , Sumoilação/genética , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Strictosidine synthase (STR), the gate enzyme for monoterpenoid indole alkaloid biosynthesis, catalyzes the Pictet-Spengler reaction (PSR) of various tryptamine derivatives with secologanin assisted by "indole sandwich" stabilization. Continuous exploration with ß-methyltryptamine (IPA) stereoselectively delivered the C6-methylstrictosidines and C6-methylvincosides by enzymatic and nonenzymatic PSR, respectively. Unexpectedly, the first "nonindole sandwich" binding mode was witnessed by the X-ray structures of STR1-ligand complexes. Site-directed mutagenesis revealed the critical cryptic role of the hydroxyl group of Tyr151 in IPA biotransformation. Further computational calculations demonstrated the adjustable IPA position in STR1 upon the binding of secologanin, and Tyr151-OH facilitates the productive PSR binding mode via an advantageous hydrogen-bond network. Further chemo-enzymatic manipulation of C6-methylvincosides successfully resulted in the discovered antimalarial framework (IC50 = 0.92 µM).
Assuntos
Alcaloides , Carbolinas , Carbono-Nitrogênio Liases , Triptaminas , Humanos , Alcaloides/química , Alcaloides/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Carbolinas/química , Carbolinas/metabolismo , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Modelos Moleculares , Estrutura Molecular , p-Hidroxianfetamina , Ligação Proteica , Conformação Proteica , Triptaminas/química , Triptaminas/metabolismoRESUMO
Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases.
Assuntos
Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/metabolismo , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Domínio Catalítico , Cisteína Endopeptidases , Humanos , Cinética , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , SumoilaçãoRESUMO
Ergothioneine is a histidine-derived sulfur metabolite that is biosynthesized by bacteria and fungi. Plants and animals absorb ergothioneine as a micronutrient from their environment or nutrition. Several different mechanisms of microbial ergothioneine production have been described in the past ten years. Much less is known about the genetic and structural basis for ergothioneine catabolism. In this report, we describe the in vitro reconstitution of a five-step pathway that degrades ergothioneine to l-glutamate, trimethylamine, hydrogen sulfide, carbon dioxide, and ammonia. The first two steps are catalyzed by the two enzymes ergothionase and thiourocanate hydratase. These enzymes are closely related to the first two enzymes in histidine catabolism. However, the crystal structure of thiourocanate hydratase from the firmicute Paenibacillus sp. reveals specific structural features that strictly differentiate the activity of this enzyme from that of urocanate hydratases. The final two steps are catalyzed by metal-dependent hydrolases that share most homology with the last two enzymes in uracil catabolism. The early and late part of this pathway are connected by an entirely new enzyme type that catalyzes desulfurization of a thiohydantoin intermediate. Homologous enzymes are encoded in many soil-dwelling firmicutes and proteobacteria, suggesting that bacterial activity may have a significant impact on the environmental availability of ergothioneine.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Carbono-Oxigênio Liases/metabolismo , Liases de Carbono-Enxofre/metabolismo , Ergotioneína/metabolismo , Hidrolases/metabolismo , Proteínas de Bactérias/química , Biocatálise , Carbono-Nitrogênio Liases/química , Carbono-Oxigênio Liases/química , Liases de Carbono-Enxofre/química , Hidrolases/química , Paenibacillus/metabolismo , Metabolismo Secundário , Especificidade por SubstratoRESUMO
Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.
Assuntos
Automação Laboratorial/métodos , Testes de Coagulação Sanguínea/métodos , Carbono-Nitrogênio Liases/metabolismo , Deficiência do Fator XIII/diagnóstico , Fator XIII/análise , Corantes Fluorescentes/normas , Automação Laboratorial/normas , Bilirrubina/metabolismo , Testes de Coagulação Sanguínea/normas , Compostos Cromogênicos/normas , Fator XIII/metabolismo , Deficiência do Fator XIII/sangue , Fluorometria/métodos , Fluorometria/normas , Hemólise , Humanos , Testes Imunológicos/métodos , Testes Imunológicos/normas , Reprodutibilidade dos Testes , Transglutaminases/metabolismoRESUMO
Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved.
Assuntos
Biocatálise , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/metabolismo , Eurotiales/enzimologia , Biodegradação Ambiental , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , TemperaturaRESUMO
YggS (COG0325) is a pyridoxal 5'-phosphate (PLP)-binding protein proposed to be involved in homeostasis of B6 vitamers. In Salmonella enterica, lack of yggS resulted in phenotypes that were distinct and others that were similar to those of a yggS mutant of Escherichia coli Like other organisms, yggS mutants of S. enterica accumulate endogenous pyridoxine 5'-phosphate (PNP). Data herein show that strains lacking YggS accumulated â¼10-fold more PLP in growth medium than a parental strain. The deoxyxylulose 5-phosphate-dependent biosynthetic pathway for PLP and the PNP/pyridoxamine 5'-phosphate (PMP) oxidase credited with interconverting B6 vitamers were replaced with a single PLP synthase from Saccharomyces cerevisiae The impact of a yggS deletion on the intracellular and extracellular levels of B6 vitamers in this restructured strain supported a role for PdxH in PLP homeostasis and led to a general model for YggS function in PLP-PMP cycling. Our findings uncovered broader consequences of a yggS mutation than previously reported and suggest that the accumulation of PNP is not a direct effect of lacking YggS but rather a downstream consequence.IMPORTANCE Pyridoxal 5'-phosphate (PLP) is an essential cofactor for enzymes in all domains of life. Perturbations in PLP or B6 vitamer content can be detrimental, notably causing B6-dependent epilepsy in humans. YggS homologs are broadly conserved and have been implicated in altered levels of B6 vitamers in multiple organisms. The biochemical activity of YggS, expected to be conserved across domains, is not yet known. Herein, a simplified heterologous pathway minimized metabolic variables and allowed the dissection of this system to generate new metabolic knowledge that will be relevant to understanding YggS.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Proteínas de Transporte/metabolismo , Fosfato de Piridoxal/análogos & derivados , Proteínas de Saccharomyces cerevisiae/metabolismo , Salmonella enterica/metabolismo , Vitamina B 6/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Carbono-Nitrogênio Liases/genética , Proteínas de Transporte/genética , Homeostase , Mutação , Fosfato de Piridoxal/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Salmonella enterica/genéticaRESUMO
Plasma transglutaminase FXIII provides mechanical and biochemical stability to blood clots. Congenital or acquired deficiency may be associated with bleeding diathesis and requires therefore careful monitoring. The precise automated measurement of a large number of plasma samples can provide new insights regarding the clinical relevance of certain FXIII levels. There is still the unmet diagnostic need for a reliable high-throughput method. Here we report the development and feasibility study of a promising prototype, adapting the precise FXIIIa isopeptidase assay principle on the optimized automated Ceveron s100 platform.
Assuntos
Automação , Carbono-Nitrogênio Liases/metabolismo , Fator XIIIa/metabolismo , Estudos de Viabilidade , Humanos , ProteóliseRESUMO
Carbon-nitrogen (C-N) lyases are enzymes that normally catalyze the cleavage of C-N bonds. Reversing this reaction towards carbon-nitrogen bond formation can be a powerful approach to prepare valuable compounds that could find applications in everyday life. This review focuses on recent (last five years) applications of native and engineered C-N lyases, either as stand-alone biocatalysts or as part of multienzymatic and chemoenzymatic cascades, in enantioselective synthesis of noncanonical amino acids and dinitrogen-fused heterocycles, which are useful tools for neurobiological research and important synthetic precursors to pharmaceuticals and food additives.
Assuntos
Aminoácidos/biossíntese , Carbono-Nitrogênio Liases/metabolismo , Compostos Heterocíclicos/metabolismo , Aminoácidos/química , Biocatálise , Compostos Heterocíclicos/química , Estrutura Molecular , EstereoisomerismoRESUMO
Blood coagulation factor XIII-A (FXIII-A), a member of the transglutaminase enzyme family, is best known for its fibrin clot stabilizing function during blood coagulation. It possesses amine incorporating and protein crosslinking transamidase activities, but it is also able to cleave the previously formed isopeptide bond by its isopeptidase activity. Our aim was to develop a protein-based assay for better characterization of FXIII-A isopeptidase activity. The first attempt applying the crosslinked D-dimer of fibrin as a substrate was not successful because of poor reproducibility. Then, the principle of an earlier published anisotropy based activity assay was adapted for the measurement of FXIII-A isopeptidase activity. After crosslinking the fluorescently labelled α2-antiplasmin derived peptide and S100A4(GST) lysine donor protein, this protease-resistant γ-glutamyl-ε-lysine isopeptide bond containing protein-peptide product was applied as a substrate for FXIII-A. Using this substrate and detecting decreasing anisotropy, kinetic measurement of FXIII-A isopeptidase activity was achieved at high sensitivity even in a complex biological sample and in the presence of inhibitor.
Assuntos
Carbono-Nitrogênio Liases/metabolismo , Fator XIIIa/metabolismo , Anisotropia , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/isolamento & purificação , Fator XIIIa/química , Fluorescência , HumanosRESUMO
The Pictet-Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-ß-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR) catalyzes the Pictet-Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept short-chain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% ee STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila (OpSTR) using a combination of X-ray crystallography, mutational, and molecular dynamics (MD) studies. We discovered that short-chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.
Assuntos
Carbono-Nitrogênio Liases/metabolismo , Catálise , Ligação Proteica , Estereoisomerismo , Especificidade por SubstratoRESUMO
Pyridoxine (vitamin B6) and its vitamers are used by living organisms both as enzymatic cofactors and as antioxidants. We used Arabidopsis pyridoxine biosynthesis mutant pdx1.3-1 to study the involvement of the PLP-synthase main polypeptide PDX1 in plant responses to ultraviolet radiation of two different qualities, one containing primarily UV-A (315-400 nm) and the other containing both UV-A and UV-B (280-315 nm). The antioxidant capacity and the flavonoid and glucosinolate (GS) profiles were examined. As an indicator of stress, Fv/Fm of photosystem II reaction centers was used. In pdx1.3-1, UV-A + B exposure led to a significant 5% decrease in Fv/Fm on the last day (day 15), indicating mild stress at this time point. The antioxidant capacity of Col-0 wildtype increased significantly (50-73%) after 1 and 3 days of UV-A + B. Instead, in pdx1.3-1, the antioxidant capacity significantly decreased by 44-52% over the same time period, proving the importance of a full complement of functional PDX1 genes for the detoxification of reactive oxygen species. There were no significant changes in the flavonoid glycoside profile under any light condition. However, the GS profile was significantly altered, both with respect to Arabidopsis accession and exposure to UV. The difference in flavonoid and GS profiles reflects that the GS biosynthesis pathway contains at least one pyridoxine-dependent enzyme, whereas no such enzyme is used in flavonoid biosynthesis. Also, there was strong correlation between the antioxidant capacity and the content of some GS compounds. Our results show that vitamin B6 vitamers, functioning both as antioxidants and co-factors, are of importance for the physiological fitness of plants.
Assuntos
Antioxidantes/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Carbono-Nitrogênio Liases/metabolismo , Glucosinolatos/biossíntese , Raios Ultravioleta , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Carbono-Nitrogênio Liases/genética , Cromatografia Líquida de Alta Pressão , Flavonoides/biossíntese , Glucosinolatos/análise , Mutagênese , Complexo de Proteína do Fotossistema II/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismoRESUMO
Experimental assessment of catalytic reaction mechanisms and profiles of radical enzymes can be severely challenging due to the reactive nature of the intermediates and sensitivity of cofactors such as iron-sulfur clusters. Here, we present an enzyme-directed computational methodology for the assessment of thermodynamic reaction profiles and screening for radical stabilization energies (RSEs) for the assessment of catalytic turnovers in radical enzymes. We have applied this new screening method to the radical S-adenosylmethione enzyme 7-carboxy-7-deazaguanine synthase (QueE), following a detailed molecular dynamics (MD) analysis that clarifies the role of both specific enzyme residues and bound Mg2+, Ca2+, or Na+. The MD simulations provided the basis for a statistical approach to sample different conformational outcomes. RSE calculation at the M06-2X/6-31+G* level of theory provided the most computationally cost-effective assessment of enzyme-based energies, facilitated by an initial triage using semiempirical methods. The impact of intermolecular interactions on RSE was clearly established, and application to the assessment of potential alternative substrates (focusing on radical clock type rearrangements) proposes a selection of carbon-substituted analogues that would react to afford cyclopropylcarbinyl radical intermediates as candidates for catalytic turnover by QueE.
Assuntos
Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Carbono-Nitrogênio Liases/química , Metais/metabolismo , Conformação ProteicaRESUMO
The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 can grow with cyanate, cyanide, or cyanide-containing industrial residues as the sole nitrogen source, but the assimilation of cyanide and cyanate takes place through independent pathways. Therefore, cyanide degradation involves a chemical reaction between cyanide and oxaloacetate to form a nitrile that is hydrolyzed to ammonium by the nitrilase NitC, whereas cyanate assimilation requires a cyanase that catalyzes cyanate decomposition to ammonium and carbon dioxide. The P. pseudoalcaligenes CECT5344 cynFABDS gene cluster codes for the putative transcriptional regulator CynF, the ABC-type cyanate transporter CynABD, and the cyanase CynS. In this study, transcriptional analysis revealed that the structural cynABDS genes constitute a single transcriptional unit, which was induced by cyanate and repressed by ammonium. Mutational characterization of the cyn genes indicated that CynF was essential for cynABDS gene expression and that nitrate/nitrite transporters may be involved in cyanate uptake, in addition to the CynABD transport system. Biodegradation of hazardous jewelry wastewater containing high amounts of cyanide and metals was achieved in a batch reactor operating at an alkaline pH after chemical treatment with hydrogen peroxide to oxidize cyanide to cyanate.
Assuntos
Proteínas de Bactérias/genética , Cianatos/metabolismo , Família Multigênica , Pseudomonas pseudoalcaligenes/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Cianetos/metabolismo , Pseudomonas pseudoalcaligenes/metabolismo , Águas Residuárias/análise , Águas Residuárias/microbiologiaRESUMO
Vitamin B6 (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B6 content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B6 in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B6 biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B6 levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B6 biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B6 in leaves (up to 28.3-fold) and roots (up to 12-fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B6 did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B6 content could also be achieved in rice seeds (up to 3.1-fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B6 -enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.
Assuntos
Arabidopsis/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Vitamina B 6/biossíntese , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Transferases de Grupos Nitrogenados/genética , Transferases de Grupos Nitrogenados/metabolismo , Oryza/genética , Oryza/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Estresse Salino/fisiologia , Sementes/metabolismo , Transgenes , Vitamina B 6/metabolismo , Xanthomonas/patogenicidadeRESUMO
Pseudoenzymes have burst into the limelight recently as they provide another dimension to regulation of cellular protein activity. In the eudicot plant lineage, the pseudoenzyme PDX1.2 and its cognate enzyme PDX1.3 interact to regulate vitamin B6 biosynthesis. This partnership is important for plant fitness during environmental stress, in particular heat stress. PDX1.2 increases the catalytic activity of PDX1.3, with an overall increase in vitamin B6 biosynthesis. However, the mechanism by which this is achieved is not known. In this study, the Arabidopsis thaliana PDX1.2-PDX1.3 complex was crystallized in the absence and presence of ligands, and attempts were made to solve the X-ray structures. Three PDX1.2-PDX1.3 complex structures are presented: the PDX1.2-PDX1.3 complex as isolated, PDX1.2-PDX1.3-intermediate (in the presence of substrates) and a catalytically inactive complex, PDX1.2-PDX1.3-K97A. Data were also collected from a crystal of a selenomethionine-substituted complex, PDX1.2-PDX1.3-SeMet. In all cases the protein complexes assemble as dodecamers, similar to the recently reported individual PDX1.3 homomer. Intriguingly, the crystals of the protein complex are statistically disordered owing to the high degree of structural similarity of the individual PDX1 proteins, such that the resulting configuration is a composite of both proteins. Despite the differential methionine content, selenomethionine substitution of the PDX1.2-PDX1.3 complex did not resolve the problem. Furthermore, a comparison of the catalytically competent complex with a noncatalytic complex did not facilitate the resolution of the individual proteins. Interestingly, another catalytic lysine in PDX1.3 (Lys165) that pivots between the two active sites in PDX1 (P1 and P2), and the corresponding glutamine (Gln169) in PDX1.2, point towards P1, which is distinctive to the initial priming for catalytic action. This state was previously only observed upon trapping PDX1.3 in a catalytically operational state, as Lys165 points towards P2 in the resting state. Overall, the study shows that the integration of PDX1.2 into a heteromeric dodecamer assembly with PDX1.3 does not cause a major structural deviation from the overall architecture of the homomeric complex. Nonetheless, the structure of the PDX1.2-PDX1.3 complex highlights enhanced flexibility in key catalytic regions for the initial steps of vitamin B6 biosynthesis. This report highlights what may be an intrinsic limitation of X-ray crystallography in the structural investigation of pseudoenzymes.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Vitamina B 6/metabolismoRESUMO
Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.
Assuntos
Vias Biossintéticas/genética , Resistência à Doença/genética , Doenças das Plantas/genética , Solanum lycopersicum/genética , Vitamina B 6/biossíntese , Antibiose , Bacillus subtilis/fisiologia , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Piridoxal Quinase/genética , Piridoxal Quinase/metabolismo , Xanthomonas vesicatoria/fisiologiaRESUMO
Covalent conjugation of the ubiquitin-related SUMO modifier to lysine residues of cellular proteins (SUMOylation) is a prevalent posttranslational modification. SUMOs are synthesized as precursor proteins that require carboxy-terminal processing prior to conjugation. Subsequently, a multistep enzymatic pathway is used for conjugation to target proteins. SUMOylation generally impacts protein-protein interactions and the assembly of multiprotein complexes. Cellular processes regulated by SUMOylation include DNA damage responses, cell cycle progression, or the control of gene expression. SUMOylation is reversible and commonly only a small fraction of a particular SUMO target is modified at a given time. Deconjugation of SUMO is catalyzed by a group of cysteine proteases termed SUMO proteases or SUMO isopeptidases. In human cells nine SUMO proteases, belonging to three separate families of cysteine proteases have been identified so far. The regulation and target specificity of individual SUMO proteases have not been dissected in detail, but the current view is that each protease controls the modification of subsets of proteins that are functionally and/or physically linked. Importantly, some SUMO proteases/isopeptidases not only function in deconjugation of SUMO from proteins, but also act in C-terminal processing of the SUMO precursors. Here we describe general methods for monitoring SUMO protease/isopeptidase activities in cell or tissue extracts.
Assuntos
Cisteína Proteases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Carbono-Nitrogênio Liases/metabolismo , Ensaios Enzimáticos/métodos , Humanos , Modelos Moleculares , Especificidade por Substrato , SumoilaçãoRESUMO
This study aimed to investigate the effects of desumoylating isopeptidase 2 (DESI2) on tumor cell proliferation, apoptosis and invasion of pancreatic cancer, and to assess the signaling pathway involved. Overexpression and silence of DESI2 were designed and the experiments were divided into 5 groups: a normal control group, an interference control group (shRNA-NC); an interference group (sh-DESI2); an overexpression control group (NC), an overexpression group (DESI2). Quantitative real time polymerase chain reaction (qRT-PCR) was used to screen the appropriate interference sequence. The silencing and overexpression of DESI2 were confirmed by qRT-PCR and western blotting. Cell cycling, apoptosis, invasion, and the expression of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway and caspase 3 were measured. Overexpression and silence of DESI2 were successfully designed in two pancreatic cancer cells, and the interference effect of sh-DESI2-3 showed the best silencing effects. The biological activities of DESI2 were detected in both ASPC-1 and PANCE-1 cells. Our results showed that cell proliferation was significantly increased in the sh-DESI2 group, while decreased in DESI2 group compared with the control group in both cell lines. In ASPC-1 cells, the events in G1 phase decreased and in S phase increased obviously in the sh-DESI2 group, compared with control group. An opposite result was found when DESI2 was overexpressed. In PANCE-1 cells, the events in G2 phase were higher in the sh-DESI2 group, while in the DESI2 group was significantly lower than that in control group. In ASPC-1 and PANCE-1 cells, sh-DESI2 group showed decreased apoptosis, increased cell invasion and increased expression of AKT, p-Akt, PI3K, p-PI3K, p-mTOR and mTOR and decreased caspase 3 expression compared with the control group, while overexpression of DESI2 leaded to increased apoptosis, decreased cell invasion and reduced expression of AKT, p-Akt, PI3K, p-PI3K, p-mTOR and mTOR and increased expression of caspase 3. DESI2 regulates the proliferation and apoptosis of pancreatic cancer cells through PI3K/AKT/mTOR signaling pathway.