RESUMO
The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.
Assuntos
Infecções por Burkholderia , Burkholderia cenocepacia , Doença Granulomatosa Crônica , Homogentisato 1,2-Dioxigenase , Melaninas , Animais , Feminino , Humanos , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/patogenicidade , Burkholderia cenocepacia/metabolismo , Infecções por Burkholderia/microbiologia , Fibrose Cística/microbiologia , Modelos Animais de Doenças , Doença Granulomatosa Crônica/microbiologia , Doença Granulomatosa Crônica/genética , Homogentisato 1,2-Dioxigenase/genética , Homogentisato 1,2-Dioxigenase/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Melaninas/metabolismo , Mutação , Estresse Oxidativo , Virulência/genéticaRESUMO
White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.
Assuntos
Dioxigenases , Phanerochaete , Lignina/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Phanerochaete/genética , Homogentisato 1,2-Dioxigenase/metabolismo , Proteínas/metabolismo , Peroxidases/genética , Peroxidases/metabolismoRESUMO
Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs, which occurs because the homogentisate 1,2-dioxygenase (HGD) enzyme is not functional due to gene variants. Over time, HGA oxidation and accumulation cause the formation of the ochronotic pigment, a deposit that provokes tissue degeneration and organ malfunction. Here, we report a comprehensive review of the variants so far reported, the structural studies on the molecular consequences of protein stability and interaction, and molecular simulations for pharmacological chaperones as protein rescuers. Moreover, evidence accumulated so far in alkaptonuria research will be re-proposed as the bases for a precision medicine approach in a rare disease.
Assuntos
Alcaptonúria , Homogentisato 1,2-Dioxigenase , Humanos , Alcaptonúria/genética , Alcaptonúria/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Estudos de Associação Genética , Homogentisato 1,2-Dioxigenase/genética , Homogentisato 1,2-Dioxigenase/metabolismo , Ácido Homogentísico/metabolismo , Doenças Raras , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Alkaptonuria is a rare autosomal recessive genetic disorder resulting from the deficiency of homogentisate 1,2 dioxygenase (HGD), the third enzyme in the tyrosine degradation pathway. Homogentisic acid produced in excess oxidizes into ochronotic pigment polymer. Accumulation of this pigment in various tissues leads to systemic disease. METHODS: Clinical, laboratory, molecular findings and treatment characteristics of 35 patients followed up in Ege University Pediatric Nutrition, and Metabolism Department with the diagnosis of alkaptonuria were evaluated retrospectively. RESULTS: Twenty-four males (68.57%) and 11 females (31.42%) with a confirmed diagnosis of alkaptonuria from 32 different families were included in the study. We identified 11 different genetic variants; six of these were novel. c.1033C>T, c.676G>A, c.664G>A, c.731_734del, c.1009G>T, c.859_862delins ATAC were not previously reported in the literature. 24 (68.57%) patients only adhered to a low-protein diet in our study group. Seven (20%) patients initiated a low protein diet and NTBC therapy. Mean urinary HGA decreased by 88.7% with nitisinone. No statistical changes were detected in urinary HGA excretion with the low protein diet group. CONCLUSIONS: In our study, alkaptonuria patients were diagnosed at different ages, from infancy to adulthood, and progressed with other systemic involvement in the follow-up. Since the initial period is asymptomatic, giving potentially effective treatment from an early age is under discussion. Raising disease awareness is very important in reducing disease mortality and morbidity rates.
Assuntos
Alcaptonúria , Adulto , Alcaptonúria/diagnóstico , Alcaptonúria/epidemiologia , Alcaptonúria/genética , Criança , Feminino , Seguimentos , Homogentisato 1,2-Dioxigenase/genética , Homogentisato 1,2-Dioxigenase/metabolismo , Ácido Homogentísico/metabolismo , Humanos , Masculino , Estudos Retrospectivos , TirosinaRESUMO
Kidney renal clear cell carcinoma (KIRC) with poor prognosis is the main histological subtype of renal cell carcinoma, accounting for more than 80% of patients. Most patients are diagnosed at an advanced stage due to being asymptomatic early on. Advanced KIRC has an extremely poor prognosis due to its inherent resistance to radiotherapy and chemotherapy. Therefore, a comprehensive understanding of the molecular mechanisms of KIRC and the development of effective early diagnostic and therapeutic strategies is urgently needed. In this study, we aimed to identify the prognosis-related biomarker and analyzed its relationship with tumor progression. Metabolic changes are an important feature of kidney cancer, where the reduction of fumarate allows us to target the tyrosine metabolic pathway. The homogentisate 1,2-dioxygenase (HGD) and glutathione S-transferase zeta 1 (GSTZ1) related with prognosis of KIRC was identified through bioinformatics analysis based on The Cancer Genome Atlas (TCGA) databases. Mechanistically, we found that decreased HGD and GSTZ1 promote aerobic glycolysis in KIRC, coordinate the balance of amino acid metabolism and energy metabolism in tumor cells, and ultimately activate the tumor cell cycle and tumor progression. In summary, we identified the tyrosine metabolizing enzymes HGD and GSTZ1 as biomarkers of KIRC, which will further the understanding of the tumor metabolism profile, provide novel strategies and theoretical support for diagnosing and treating KIRC and as referential for future clinical research.
Assuntos
Carcinoma de Células Renais , Glutationa Transferase , Homogentisato 1,2-Dioxigenase , Neoplasias Renais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Dioxigenases/sangue , Dioxigenases/metabolismo , Feminino , Glutationa Transferase/sangue , Glutationa Transferase/metabolismo , Homogentisato 1,2-Dioxigenase/sangue , Homogentisato 1,2-Dioxigenase/metabolismo , Humanos , Rim/metabolismo , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Tirosina/metabolismoRESUMO
Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.
Assuntos
Alcaptonúria/prevenção & controle , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Homogentisato 1,2-Dioxigenase/metabolismo , Ácido Homogentísico/metabolismo , Alcaptonúria/metabolismo , Apoptose/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Linhagem Celular , Condrócitos/citologia , Ácido Homogentísico/farmacologia , Humanos , Ocronose/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de SinaisRESUMO
Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.
Assuntos
Acetatos/urina , Alcaptonúria/urina , Benzoquinonas/urina , Homogentisato 1,2-Dioxigenase/metabolismo , Ácido Homogentísico/urina , Ocronose/metabolismo , Ocronose/urina , Adulto , Idoso , Alcaptonúria/enzimologia , Alcaptonúria/genética , Estudos de Casos e Controles , Difusão Dinâmica da Luz , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Homogentisato 1,2-Dioxigenase/genética , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Ocronose/enzimologia , Ocronose/genética , Oxirredução , Espectrofotometria Ultravioleta , UrináliseRESUMO
Alkaptonuria is a rare genetic disease caused by mutations in HGD gene. Here we report the results of genetic and biochemical analysis of 49 Russian patients with alkaptonuria. One of the common variants c.481G > A; p.(Gly161Arg) comprising 72.4% of identified alleles was found in 45 of 49 patients in our cohort, which is probably the highest frequency of this variant worldwide. 9 novel variants were found: 6 missense, 2 splicing and 1 loss of start-codon. For missense variants we performed bioinformatic analysis, protein 3D-modeling and molecular dynamics simulations, which strongly suggest their pathogenic effect. For the rare synonymous variant c.753C > T; p.(Gly251Gly), which was found in 3 cases and predicted to activate cryptic splice site, we performed the detailed functional analysis on patient's cDNA and minigene assay and confirmed its pathogenicity.
Assuntos
Alcaptonúria/genética , Homogentisato 1,2-Dioxigenase/genética , Mutação , Frequência do Gene , Células Hep G2 , Homogentisato 1,2-Dioxigenase/química , Homogentisato 1,2-Dioxigenase/metabolismo , Humanos , Simulação de Dinâmica Molecular , Sítios de Splice de RNARESUMO
Alkaptonuria (AKU) is an ultra-rare disease caused by mutations in homogentisate 1,2-dioxygenase (HGD) enzyme, characterized by the loss of enzymatic activity and the accumulation of its substrate, homogentisic acid (HGA) in different tissues, leading to ochronosis and organ degeneration. Although the pathological effects of HGD mutations are largely studied, less is known about the structure of the enzyme, in particular the pathways for dioxygen diffusion to the active site, required for the enzymatic reaction, are still uninvestigated. In the present project, the combination of two in silico techniques, Molecular Dynamics (MD) simulation and Implicit Ligand Sampling (ILS), was used to delineate gas diffusion routes in HGD enzyme. A route from the central opening of the hexameric structure of the enzyme to the back of the active site trough the protein moiety was identified as the path for dioxygen diffusion, also overlapping with a transient pocket, which then assumes an important role in dioxygen diffusion. Along the route the sequence location of the missense variant E401Q, responsible for AKU development, was also found, suggesting such mutation to be conducive of enzymatic activity loss by altering the flow dynamics of dioxygen. Our in silico approach allowed also to delineate the route of HGA substrate to the active site, until now only supposed.
Assuntos
Alcaptonúria/patologia , Dioxigenases/metabolismo , Homogentisato 1,2-Dioxigenase/metabolismo , Alcaptonúria/enzimologia , Cristalografia por Raios X , Difusão , Dioxigenases/química , Homogentisato 1,2-Dioxigenase/química , Humanos , Modelos Moleculares , Conformação Proteica , TermodinâmicaRESUMO
Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.
Assuntos
Alcaptonúria/enzimologia , Homogentisato 1,2-Dioxigenase/genética , Ácido Homogentísico/metabolismo , Fígado/enzimologia , Alcaptonúria/genética , Alcaptonúria/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Homogentisato 1,2-Dioxigenase/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras GenéticasRESUMO
Bacillus thuringiensis BMB181 (Bt BMB181), with high melanin production, is an ideal candidate for industrial scale production of light-stable insecticides. However, its melanogenic pathways remain unclear. In the present study, we demonstrated that Bt BMB181 failed to produce melanin after treatment with mesotrione, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the homogentisic acid pathway of melanin synthesis. Heterologous expression experiments suggested that homogentisate-1,2-dioxygenase (HmgA) in Bt BMB171 functions normally, yet HmgA in Bt BMB181 had lost its activity, at least partly. Using the CRISPR-Cas9 system, the hmgA gene in Bt BMB171 was knocked out and the mutant strain gained the ability to produce melanin. Furthermore, the complemented strain reverted to the wild-type phenotype. In addition, overexpression of its own hmgA gene in Bt BMB181 also resulted in failure to produce the pigment. BLAST results indicated that the amino acid alteration (G272E) in HmgA of Bt BMB181 was caused by a single point mutation (815Gâ A). The enzyme activity of purified HmgA171 was more than 10-fold higher than that of HmgA181. Finally, we determined that the mutation in hmgA was responsible for melanin accumulation in Bt BMB181. Our results provided new insights into the synthesis and regulation of melanin production in B.thuringiensis and will promote its future industrial application.
Assuntos
Bacillus thuringiensis/enzimologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Homogentisato 1,2-Dioxigenase/metabolismo , Melaninas/metabolismo , Mutação Puntual , Bacillus thuringiensis/genética , Bacillus thuringiensis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Homogentisato 1,2-Dioxigenase/genéticaRESUMO
Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaverâ»Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.
Assuntos
Proteínas de Bactérias/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Pseudomonas chlororaphis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Catecol 1,2-Dioxigenase/química , Catecol 1,2-Dioxigenase/classificação , Catecóis/metabolismo , Clorofenóis/química , Clorofenóis/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Homogentisato 1,2-Dioxigenase/química , Homogentisato 1,2-Dioxigenase/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Metais/química , Metais/metabolismo , Filogenia , Estabilidade Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
MAIN CONCLUSION: Fumarylacetoacetate hydrolase participates in positive regulation of salt stress in Arabidopsis. Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolysis of fumarylacetoacetate into fumarate and acetoacetate, the final step in the Tyr degradation pathway that is essential to animals. However, the Tyr degradation pathway is not well understood in plants. Previously, we found that mutation of the SHORT-DAY SENSITIVE CELL DEATH 1 (SSCD1) gene encoding FAH in Arabidopsis causes spontaneous cell death under short day, which first indicated that the Tyr degradation pathway also plays an important role in plants. In this study, we found that the SSCD1 gene was up-regulated by salt stress, and the sscd1 mutant was hypersensitive to salt stress. However, the double mutant of SSCD1 and HOMOGENTISATE DIOXYGENASE, in which intermediates of the Tyr degradation pathway could not be produced, displayed a normal response to salt stress. Furthermore, the sscd1 mutant showed more accumulation of reactive oxygen species (ROS) and less up-regulation of some ROS-scavenging genes such as ASCORBATE PEROXIDASE 2 and COPPER/ZINC SUPEROXIDE DISMUTASE 1 compared with wild type under salt stress. In addition, SSCD1 expression was also up-regulated by H2O2, and the sscd1 mutant exhibited hypersensitivity to oxidative stress compared with wild type. Taken together, we concluded that loss of FAH in sscd1 leads to the accumulation of Tyr degradation intermediates, which impairs the up-regulation of some ROS-scavenging genes under salt stress, causing more accumulation of ROS, resulting in the hypersensitivity of sscd1 to salt stress.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ascorbato Peroxidases/metabolismo , Morte Celular , Homogentisato 1,2-Dioxigenase/metabolismo , Peróxido de Hidrogênio/metabolismo , Hidrolases/genética , Mutação , Estresse Oxidativo , Tolerância ao Sal , Regulação para CimaRESUMO
Protozoan predation is one of the main environmental factors constraining bacterial growth in aquatic environments, and thus has led to the evolution of a number of defence mechanisms that protect bacteria from predation. These mechanisms may also function as virulence factors in infection of animal and human hosts. Whole transcriptome shotgun sequencing of Vibrio cholerae biofilms during predation by the amoebae, Acanthamoeba castellanii, revealed that 131 transcripts were significantly differentially regulated when compared to the non-grazed control. Differentially regulated transcripts included those involved in biosynthetic and metabolic pathways. The transcripts of genes involved in tyrosine metabolism were down-regulated in the grazed population, which indicates that the tyrosine metabolic regulon may have a role in the response of V. cholerae biofilms to A. castellanii predation. Homogentisate 1, 2-dioxygenase (HGA) is the main intermediate of the normal L-tyrosine catabolic pathway which is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in hmgA, was more resistant to amoebae predation than the wild type. Increased grazing resistance was correlated with increased production of pyomelanin and thus reactive oxygen species (ROS), suggesting that ROS production is a defensive mechanism used by bacterial biofilms against predation by amoebae A. castellanii.
Assuntos
Acanthamoeba castellanii/microbiologia , Antiprotozoários/metabolismo , Biofilmes/crescimento & desenvolvimento , Melaninas/metabolismo , Vibrio cholerae/crescimento & desenvolvimento , Animais , Homogentisato 1,2-Dioxigenase/genética , Homogentisato 1,2-Dioxigenase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Vibrio cholerae/genética , Fatores de VirulênciaRESUMO
Alkaptonuria (AKU) is an inborn error of metabolism where mutation of homogentisate 1,2-dioxygenase (HGD) gene leads to a deleterious or misfolded product with subsequent loss of enzymatic degradation of homogentisic acid (HGA) whose accumulation in tissues causes ochronosis and degeneration. There is no licensed therapy for AKU. Many missense mutations have been individuated as responsible for quaternary structure disruption of the native hexameric HGD. A new approach to the treatment of AKU is here proposed aiming to totally or partially rescue enzyme activity by targeting of HGD with pharmacological chaperones, i.e. small molecules helping structural stability. Co-factor pockets from oligomeric proteins have already been successfully exploited as targets for such a strategy, but no similar sites are present at HGD surface; hence, transient pockets are here proposed as a target for pharmacological chaperones. Transient pockets are detected along the molecular dynamics trajectory of the protein and filtered down to a set of suitable sites for structural stabilization by mean of biochemical and pharmacological criteria. The result is a computational workflow relevant to other inborn errors of metabolism requiring rescue of oligomeric, misfolded enzymes.
Assuntos
Alcaptonúria/enzimologia , Biologia Computacional , Homogentisato 1,2-Dioxigenase/metabolismo , Simulação de Dinâmica Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Alcaptonúria/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Homogentisato 1,2-Dioxigenase/química , Homogentisato 1,2-Dioxigenase/genética , Humanos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
BACKGROUND: Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named 'ochronosis' and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis. METHODS: Gel electrophoresis, Western Blot, Congo Red-based assays and electron microscopy were used to investigate the effects of HGA on the aggregation and fibrillation propensity of amyloidogenic proteins and peptides [Aß(1-42), transthyretin, atrial natriuretic peptide, α-synuclein and SAA]. LC/MS and in silico analyses were undertaken to identify possible binding sites for HGA (or its oxidative metabolite, a benzoquinone acetate or BQA) in SAA. RESULTS: We found that HGA might act as an amyloid aggregation enhancer in vitro for all the tested proteins and peptides in a time- and dose- dependent fashion, and identified a small crevice at the interface between two HGD subunits as a candidate binding site for HGA/BQA. CONCLUSIONS: HGA might be an important amyloid co- component playing significant roles in AKU amyloidosis. GENERAL SIGNIFICANCE: Our results provide a possible explanation for the clinically verified onset of amyloidotic processes in AKU and might lay the basis to setup proper pharmacological approaches to alkaptonuric ochronosis, which are still lacking.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Ácido Homogentísico/farmacologia , Agregação Patológica de Proteínas/induzido quimicamente , Alcaptonúria/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Fator Natriurético Atrial/metabolismo , Sítios de Ligação/efeitos dos fármacos , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/metabolismo , Homogentisato 1,2-Dioxigenase/metabolismo , Humanos , Ocronose/metabolismo , Oxirredução/efeitos dos fármacos , Pré-Albumina/metabolismo , Proteína Amiloide A Sérica/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.
Assuntos
Biofortificação , Glycine max/enzimologia , Homogentisato 1,2-Dioxigenase/metabolismo , Óleos de Plantas/metabolismo , Sementes/enzimologia , Vitamina E/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Arabidopsis/genética , Inibidores Enzimáticos/toxicidade , Deleção de Genes , Genoma de Planta , Herbicidas/toxicidade , Ácido Homogentísico/metabolismo , Isoenzimas/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Mutação/genética , Fenótipo , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Glycine max/efeitos dos fármacos , Glycine max/fisiologiaRESUMO
We investigated the function of 1,4-benzoquinone reductase (BQR)- and homogentisate 1,2-dioxygenase (HGD)-like genes in wood degradation by Phanerochaete sordida YK-624, which exhibits high ligninolytic activity and selectivity. We determined homologous expression in the genomic and cDNA sequences of BQR- and HGD-like genes in P. sordida YK-624 (PsBQR and PsHGD). Both genes shared high homology (≥90 % amino acid sequence similarity) with the corresponding genes in Phanerochaete chrysosporium. These genes were co-transformed with a reporter gene into an uracil auxotrophic mutant of P. sordida YK-624. The PsBQR and PsHGD co-transformants exhibited lower holocellulolytic activity and higher ligninolytic selectivity than the control transformants. In liquid culture with vanillin, both co-transformants significantly accelerated vanillin degradation. Thus, we suggest that the rapid metabolism of low-molecular weight lignin fragments, due to the homologous expression of BQR- and HGD-like genes, affects quinone redox cycling to produce hydroxyl radicals, thereby decreasing holocellulose degradation and increasing ligninolytic selectivity.
Assuntos
Proteínas Fúngicas/genética , Homogentisato 1,2-Dioxigenase/genética , Lignina/metabolismo , Phanerochaete/enzimologia , Phanerochaete/genética , Quinona Redutases/genética , Benzaldeídos/metabolismo , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Homogentisato 1,2-Dioxigenase/metabolismo , Phanerochaete/metabolismo , Quinona Redutases/metabolismo , Transformação Genética , Madeira/metabolismo , Madeira/microbiologiaRESUMO
MAIN CONCLUSION: Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.
Assuntos
Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Morte Celular , Hidrolases/metabolismo , Sacarose/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Heptanoatos , Homogentisato 1,2-Dioxigenase/metabolismo , Plântula/metabolismo , Regulação para Cima , beta-Frutofuranosidase/metabolismo , cis-trans-Isomerases/metabolismoRESUMO
To elucidate the reaction mechanism of the ring cleavage of homogentisate by homogentisate dioxygenase, quantum mechanical/molecular mechanical (QM/MM) calculations were carried out by using two systems in different protonation states of the substrate C2 hydroxyl group. When the substrate C2 hydroxyl group is ionized (the ionized pathway), the superoxo attack on the substrate is the rate-limiting step in the catalytic cycle, with a barrier of 15.9 kcal/mol. Glu396 was found to play an important role in stabilizing the bridge species and its O-O cleavage product by donating a proton via a hydrogen-bonded water molecule. When the substrate C2 hydroxyl group is not ionized (the nonionized pathway), the O-O bond cleavage of the bridge species is the rate-limiting step, with a barrier of 15.3 kcal/mol. The QM/MM-optimized geometries for the dioxygen and alkylperoxo complexes using the nonionized model (for the C2 hydroxyl group) are in agreement with the experimental crystal structures, suggesting that the C2 hydroxyl group is more likely to be nonionized.