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1.
Biochem Biophys Res Commun ; 643: 105-110, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36592583

RESUMO

The 3'-phosphoadenosine-5'-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.


Assuntos
Archaeoglobus fulgidus , Fosfotransferases (Aceptor do Grupo Álcool) , Archaeoglobus fulgidus/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sulfato Adenililtransferase/química , Adenosina Fosfossulfato/química , Adenosina Fosfossulfato/metabolismo , Fosfoadenosina Fosfossulfato , Sulfatos/metabolismo , Trifosfato de Adenosina/metabolismo
2.
Genes (Basel) ; 13(11)2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36421772

RESUMO

BACKGROUND: Brachyolmia is a skeletal disorder with an autosomal mode of inheritance (both dominant and recessive) in which the patients have a short height, scoliosis and a reduced trunk size. METHODS: From the Muzaffargarh District in Pakistan, a consanguineous family with multiple Brachyolmia-affected subjects were enrolled in the present study. Basic epidemiological data and radiographs were collected for the subjects. Whole exome sequencing (WES) which was followed by Sanger sequencing was applied to report the geneticbasic of Brachyolmia. RESULTS: The WES identified a missense mutation (c.1037 G > C, p. R346P) in exon 9 of the PAPSS2 gene that was confirmed by the Sanger sequencing in the enrolled subjects. The mutation followed a Mendalian pattern with an autosomal recessive inheritance mode. Multiple sequence alignment by Clustal Omega indicated that the PAPSS2 mutation-containing domain is highly conserved. The HEK293T whole-cell extract that was transfected with the Myc-tagged PCMV6-PAPSS2 of both the wild and mutant constructs were resolved by SDS-PAGE as well as by a Western blot, which confirmed that there are different PAPSS2 protein expression patterns when they were compared between the control and Brachyolmia patients. This difference between the normal and mutated protein was not evident when the three-dimensional computational structures were generated using homology modeling. CONCLUSION: We report a missense mutation (c.1037 G > C, p. R346P) in the PAPSS2 gene that caused Brachyolmia in a consanguineous Pakistani family.


Assuntos
Mutação de Sentido Incorreto , Osteocondrodisplasias , Humanos , Consanguinidade , Células HEK293 , Osteocondrodisplasias/genética , Sulfato Adenililtransferase/genética
3.
BMC Plant Biol ; 22(1): 491, 2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36253724

RESUMO

BACKGROUND: ATP sulfurylase (ATPS) is a crucial enzyme for the selenate assimilation pathway in plants. RESULTS: In this study, genome-wide and comparative analyses of ATPS in Cardamine hupingshanensis, including sequence and structural analyses, were performed. The expression of ChATPS gene family members in C. hupingshanensis under selenium (Se) stress was also investigated, and our results suggest that ChATPS1-2 play key roles in the response to Se stress. Nine ATPS genes were found from C. hupingshanensis, which share highly conserved sequences with ATPS from Arabidopsis thaliana. In addition, we performed molecular docking of ATP sulfurylase in complex with compounds ATP, selenate, selenite, sulfate, and sulfite. ChAPS3-1 was found to have stronger binding energies with all compounds tested. Among these complexes, amino acid residues Arg, Gly, Ser, Glu, and Asn were commonly present. CONCLUSION: Our study reveals the molecular mechanism of C. hupingshanensis ATP sulfurylase interacting with selenate, which is essential for understanding selenium assimilation. This information will guide further studies on the function of the ChATPS gene family in the selenium stress response and lay the foundation for the selenium metabolic pathway in higher plants.


Assuntos
Arabidopsis , Cardamine , Selênio , Trifosfato de Adenosina , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Cardamine/metabolismo , Simulação de Acoplamento Molecular , Ácido Selênico , Ácido Selenioso/metabolismo , Selênio/metabolismo , Sulfato Adenililtransferase/química , Sulfato Adenililtransferase/genética , Sulfato Adenililtransferase/metabolismo , Sulfatos/metabolismo , Sulfitos/metabolismo
4.
Nat Microbiol ; 7(9): 1404-1418, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35982310

RESUMO

Members of the human gut microbiome enzymatically process many bioactive molecules in the gastrointestinal tract. Most gut bacterial modifications characterized so far are hydrolytic or reductive in nature. Here we report that abundant human gut bacteria from the phylum Bacteroidetes perform conjugative modifications by selectively sulfonating steroidal metabolites. While sulfonation is a ubiquitous biochemical modification, this activity has not yet been characterized in gut microbes. Using genetic and biochemical approaches, we identify a widespread biosynthetic gene cluster that encodes both a sulfotransferase (BtSULT, BT0416) and enzymes that synthesize the sulfonate donor adenosine 3'-phosphate-5'-phosphosulfate (PAPS), including an APS kinase (CysC, BT0413) and an ATP sulfurylase (CysD and CysN, BT0414-BT0415). BtSULT selectively sulfonates steroidal metabolites with a flat A/B ring fusion, including cholesterol. Germ-free mice monocolonized with Bacteroides thetaiotaomicron ΔBT0416 exhibited reduced gastrointestinal levels of cholesterol sulfate (Ch-S) compared with wild-type B. thetaiotaomicron-colonized mice. The presence of BtSULT and BtSULT homologues in bacteria inhibited leucocyte migration in vitro and in vivo, and abundances of cluster genes were significantly reduced in patients with inflammatory bowel disease. Together, these data provide a mechanism by which gut bacteria sulfonate steroidal metabolites and suggest that these compounds can modulate immune cell trafficking in the host.


Assuntos
Bacteroides thetaiotaomicron , Vias Biossintéticas , Animais , Bactérias , Trato Gastrointestinal , Humanos , Camundongos , Sulfato Adenililtransferase
5.
Mar Drugs ; 20(7)2022 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-35877729

RESUMO

The synthesis of cell-wall sulfated galactans proceeds through UDP galactose, a major nucleotide sugar in red seaweed, whilst sulfate is transported through S-transporters into algae. Moreover, synthesis of ethylene, a volatile plant growth regulator that plays an important role in red seaweed reproduction, occurs through S-adenosyl methionine. This means that sulfur metabolism is involved in reproduction events as well as sulfated galactan synthesis of red seaweed. In this work we study the effects of methionine and MgSO4 on gene expression of polygalactan synthesis through phosphoglucomutase (PGM) and galactose 1 phosphate uridyltransferase (GALT) and of sulfate assimilation (S-transporter and sulfate adenylyltransferase, SAT) using treatment of ethylene for 15 min, which elicited cystocarp development in Grateloupia imbricata. Also, expressions of carbohydrate sulfotransferase and galactose-6-sulfurylase in charge of the addition and removal of sulfate groups to galactans backbone were examined. Outstanding results occurred in the presence of methionine, which provoked an increment in transcript number of genes encoding S-transporter and assimilation compared to controls regardless of the development stage of thalli. Otherwise, methionine diminished the transcript levels of PGM and GALT and expressions are associated with the fertilization stage of thalli of G. imbricata. As opposite, methionine and MgSO4 did not affect the transcript number of carbohydrate sulfotransferase and galactose-6-sulfurylase. Nonetheless, differential expression was obtained for sulfurylases according to the development stages of thalli of G. imbricata.


Assuntos
Rodófitas , Alga Marinha , Carragenina , Etilenos/metabolismo , Galactanos , Galactose , Metionina , Rodófitas/metabolismo , Alga Marinha/metabolismo , Sulfato Adenililtransferase , Sulfatos
6.
Mol Genet Genomic Med ; 10(5): e1916, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35261200

RESUMO

BACKGROUND: Spondyloepi(meta)physeal dysplasias (SE[M]D) are a group of inherited skeletal disorders that mainly affect bone and cartilage, and next-generation sequencing has aided the detection of genetic defects of such diseases. In this study, we aimed to identify causative variants in four Chinese families associated with SE(M)D. METHODS: We recruited four unrelated Chinese families all displaying short stature and growth retardation. Clinical manifestations and X-ray imaging were recorded for all patients. Candidate variants were identified by whole-exome sequencing (WES) and verified by Sanger sequencing. Pathogenicity was assessed by conservation analysis, 3D protein modeling and in silico prediction, and was confirmed according to American College of Medical Genetics and Genomics. RESULTS: Three novel SE(M)D-related variants c.1090dupG, c.7168 T > G, and c.2947G > C in ACAN, and one reported variant c.712C > T in PAPSS2 were identified. Among them, c.1090dupG in ACAN and c.712C > T in PAPSS2 caused truncated protein and the other two variants led to amino acid alterations. Conservation analysis revealed sites of the two missense variants were highly conserved, and bioinformatic findings confirmed their pathogenicity. 3D modeling of mutant protein encoded by c.7168 T > G(p.Trp2390Gly) in ACAN proved the structural alteration in protein level. CONCLUSION: Our data suggested ACAN is a common pathogenic gene of SE(M)D. This study enriched the genetic background of skeletal dysplasias, and expanded the mutation spectra of ACAN and PAPSS2.


Assuntos
Agrecanas , Nanismo , Complexos Multienzimáticos , Sulfato Adenililtransferase , Agrecanas/genética , China , Nanismo/genética , Humanos , Complexos Multienzimáticos/genética , Sulfato Adenililtransferase/genética
7.
J Clin Endocrinol Metab ; 107(1): 109-118, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34498693

RESUMO

PURPOSE: Generalized arterial calcification of infancy, pseudoxanthoma elasticum, autosomal recessive hypophosphatemic rickets type 2, and hypophosphatasia are rare inherited disorders associated with altered plasma levels of inorganic pyrophosphate (PPi). In this study, we aimed to establish a reference range for plasma PPi in the pediatric population, which would be essential to support its use as a biomarker in children with mineralization disorders. METHODS: Plasma samples were collected from 200 children aged 1 day to 18 years who underwent blood testing for medical conditions not affecting plasma PPi levels. PPi was measured in proband plasma utilizing a validated adenosine triphosphate (ATP) sulfurylase method. RESULTS: The analytical sensitivity of the ATP sulfurylase assay consisted of 0.15 to 10 µM PPi. Inter- and intra-assay coefficients of variability on identical samples were below 10%. The standard range of PPi in the blood plasma of children and adolescents aged 0 to 18 years was calculated as 2.36 to 4.44 µM, with a median of 3.17 µM, with no difference between male and female probands. PPi plasma levels did not differ significantly in different pediatric age groups. MAIN CONCLUSIONS: Our results yielded no noteworthy discrepancy to the reported standard range of plasma PPi in adults (2-5 µM). We propose the described ATP sulfurylase method as a diagnostic tool to measure PPi levels in plasma as a biomarker in the pediatric population.


Assuntos
Raquitismo Hipofosfatêmico Familiar/diagnóstico , Hipofosfatasia/diagnóstico , Fosfatos/sangue , Pseudoxantoma Elástico/diagnóstico , Doenças Raras/diagnóstico , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomarcadores/sangue , Criança , Pré-Escolar , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Raquitismo Hipofosfatêmico Familiar/sangue , Raquitismo Hipofosfatêmico Familiar/genética , Feminino , Humanos , Hipofosfatasia/sangue , Hipofosfatasia/genética , Lactente , Recém-Nascido , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pseudoxantoma Elástico/sangue , Pseudoxantoma Elástico/genética , Pirofosfatases/genética , Pirofosfatases/metabolismo , Doenças Raras/sangue , Doenças Raras/genética , Valores de Referência , Sulfato Adenililtransferase/metabolismo
8.
Biochem Biophys Res Commun ; 586: 1-7, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34818583

RESUMO

Sulfation is an essential modification on biomolecules in living cells, and 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is its unique and universal sulfate donor. Human PAPS synthases (PAPSS1 and 2) are the only enzymes that catalyze PAPS production from inorganic sulfate. Unexpectedly, PAPSS1 and PAPSS2 do not functional complement with each other, and abnormal function of PAPSS2 but not PAPSS1 leads to numerous human diseases including bone development diseases, hormone disorder and cancers. Here, we reported the crystal structures of ATP-sulfurylase domain of human PAPSS2 (ATPS2) and ATPS2 in complex with is product 5'-phosphosulfate (APS). We demonstrated that ATPS2 recognizes the substrates by using family conserved residues located on the HXXH and PP motifs, and achieves substrate binding and releasing by employing a non-conserved phenylalanine (Phe550) through a never observed flipping mechanism. Our discovery provides additional information to better understand the biological function of PAPSS2 especially in tumorigenesis, and may facilitate the drug discovery against this enzyme.


Assuntos
Trifosfato de Adenosina/química , Complexos Multienzimáticos/química , Proteínas de Neoplasias/química , Fosfoadenosina Fosfossulfato/química , Sulfato Adenililtransferase/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Sulfato Adenililtransferase/genética , Sulfato Adenililtransferase/metabolismo , Termodinâmica
9.
Clin Transl Med ; 11(7): e498, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34323415

RESUMO

BACKGROUND: Cervical cancer (CC) is the second leading cause of cancer death among women worldwide. Epigenetic regulation of gene expression through DNA methylation and hydroxymethylation plays a pivotal role during tumorigenesis. In this study, to analyze the epigenomic landscape and identify potential biomarkers for CCs, we selected a series of samples from normal to cervical intra-epithelial neoplasia (CINs) to CCs and performed an integrative analysis of whole-genome bisulfite sequencing (WGBS-seq), oxidative WGBS, RNA-seq, and external histone modifications profiling data. RESULTS: In the development and progression of CC, there were genome-wide hypo-methylation and hypo-hydroxymethylation, accompanied by local hyper-methylation and hyper-hydroxymethylation. Hydroxymethylation prefers to distribute in the CpG islands and CpG shores, as displayed a trend of gradual decline from health to CIN2, while a trend of increase from CIN3 to CC. The differentially methylated and hydroxymethylated region-associated genes both enriched in Hippo and other cancer-related signaling pathways that drive cervical carcinogenesis. Furthermore, we identified eight novel differentially methylated/hydroxymethylated-associated genes (DES, MAL, MTIF2, PIP5K1A, RPS6KA6, ANGEL2, MPP, and PAPSS2) significantly correlated with the overall survival of CC. In addition, no any correlation was observed between methylation or hydroxymethylation levels and somatic copy number variations in CINs and CCs. CONCLUSION: Our current study systematically delineates the map of methylome and hydroxymethylome from CINs to CC, and some differentially methylated/hydroxymethylated-associated genes can be used as the potential epigenetic biomarkers in CC prognosis.


Assuntos
Metilação de DNA , Displasia do Colo do Útero/genética , Neoplasias do Colo do Útero/genética , Biomarcadores Tumorais/genética , Ilhas de CpG , Variações do Número de Cópias de DNA , Epigenômica , Exorribonucleases/genética , Feminino , Histonas/genética , Histonas/metabolismo , Humanos , Complexos Multienzimáticos/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Transdução de Sinais , Sulfato Adenililtransferase/genética , Taxa de Sobrevida , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/mortalidade , Displasia do Colo do Útero/patologia
10.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34039816

RESUMO

We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains-Synechocystis sp. PCC6803 and Synechococcus sp. WH7803-were grown in conventional media, and media with low ammonium, low sulfate and a high CO2/low O2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.


Assuntos
Proteínas de Bactérias/metabolismo , Sulfato Adenililtransferase/metabolismo , Synechococcus/enzimologia , Synechococcus/crescimento & desenvolvimento , Synechocystis/enzimologia , Synechocystis/crescimento & desenvolvimento , Compostos de Amônio/metabolismo , Proteínas de Bactérias/genética , Sulfato Adenililtransferase/genética , Sulfatos/metabolismo , Synechococcus/genética , Synechocystis/genética
11.
Gastroenterology ; 161(1): 271-286.e11, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33819483

RESUMO

BACKGROUND & AIMS: Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. METHODS: Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. RESULTS: The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.


Assuntos
Neoplasias Associadas a Colite/prevenção & controle , Colite/prevenção & controle , Colo/enzimologia , Mucosa Intestinal/enzimologia , Mucinas/metabolismo , Complexos Multienzimáticos/metabolismo , Sulfato Adenililtransferase/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Colite/enzimologia , Colite/genética , Colite/patologia , Neoplasias Associadas a Colite/enzimologia , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/patologia , Colo/patologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/patologia , Metaboloma , Metabolômica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos/genética , Prognóstico , Receptores Citoplasmáticos e Nucleares/metabolismo , Sulfato Adenililtransferase/genética
12.
Sci Rep ; 10(1): 14989, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929147

RESUMO

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the ß-subunit of ß-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.


Assuntos
Aminoácidos/metabolismo , Antígenos de Plantas/metabolismo , Globulinas/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Proteínas de Soja/metabolismo , Soja/metabolismo , Sulfato Adenililtransferase/genética , Inibidor da Tripsina de Soja de Bowman-Birk/metabolismo , Aminoácidos Sulfúricos/genética , Aminoácidos Sulfúricos/metabolismo , Antígenos de Plantas/genética , Regulação da Expressão Gênica de Plantas , Globulinas/genética , Plantas Geneticamente Modificadas , Proteínas de Armazenamento de Sementes/genética , Sementes/genética , Sementes/metabolismo , Proteínas de Soja/genética , Soja/genética , Sulfato Adenililtransferase/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/genética
13.
Appl Microbiol Biotechnol ; 104(16): 7067-7078, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32601738

RESUMO

The synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the action sulfotransferases using the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Three enzymes are essential for the in vitro synthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5'-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS). The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biology tool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different location enable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1), ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400-1380 mg/L with accumulation of 34-66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, were used for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupled with a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing the enzyme ASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153 mg/L enzyme with accumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach to optimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be applied towards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated an autoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV). KEY POINTS : • ePathBrick used to optimize expression levels of enzymes. • Protocols have been used for the production of recombinant enzymes. • High cell density fed-batch fermentations with high yields of soluble enzymes. • Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.


Assuntos
Bactérias/metabolismo , Engenharia Metabólica/métodos , Fosfoadenosina Fosfossulfato/biossíntese , Animais , Arilsulfotransferase/genética , Bactérias/genética , Técnicas de Cultura Celular por Lotes , Fermentação , Vetores Genéticos , Cinética , Fosfoadenosina Fosfossulfato/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pirofosfatases/metabolismo , Ratos , Proteínas Recombinantes/biossíntese , Sulfato Adenililtransferase/metabolismo , Biologia Sintética/métodos
14.
Curr Genet ; 66(4): 765-774, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32125494

RESUMO

Pyricularia oryzae is the causal agent of blast disease on staple gramineous crops. Sulphur is an essential element for the biosynthesis of cysteine and methionine in fungi. Here, we targeted the P. oryzae PoMET3 encoding the enzyme ATP sulfurylase, and PoMET14 encoding the APS (adenosine-5'-phosphosulphate) kinase that are involved in sulfate assimilation and sulphur-containing amino acids biosynthesis. In P. oryzae, deletion of PoMET3 or PoMET14 separately results in defects of conidiophore formation, significant impairments in conidiation, methionine and cysteine auxotrophy, limited invasive hypha extension, and remarkably reduced virulence on rice and barley. Furthermore, the defects of the null mutants could be restored by supplementing with exogenous cysteine or methionine. Our study explored the biological functions of sulfur assimilation and sulphur-containing amino acids biosynthesis in P. oryzae.


Assuntos
Ascomicetos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sulfato Adenililtransferase/metabolismo , Ascomicetos/efeitos dos fármacos , Cisteína/metabolismo , Cisteína/farmacologia , Deleção de Genes , Hordeum/microbiologia , Hifas/patogenicidade , Hifas/fisiologia , Metionina/metabolismo , Metionina/farmacologia , Mutação , Oryza/microbiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Doenças das Plantas/microbiologia , Esporos Fúngicos , Sulfato Adenililtransferase/genética , Virulência
15.
Ecotoxicol Environ Saf ; 194: 110402, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32151867

RESUMO

Sulfur (S) application in pakchoi (Brassica chinensis L.) cultivation is vital for reducing cadmium (Cd) accumulation in the plants. However, the mechanism of S application on Cd uptake and translocation in pakchoi is unclear. In this study, a hydroponic experiment was performed to investigate the effects of S application on Cd accumulation in pakchoi at one Cd concentration (50 µM, in comparison to the control condition, 0 µM) and three S levels (0, 2, 4 mM). The results showed that excessive S application (4 mM) reduced Cd accumulation and alleviated pakchoi growth inhibition caused by Cd stress in shoots and roots. With increased S application, the proportion of Cd in the vacuolar fraction and the proportion of NaCl-extractable Cd increased in roots. Additionally, S application increased the content of glutathione (GSH) and phytochelatins (PCs). The reduced Cd uptake and accumulation in pakchoi shoots could have been due to increased Cd chelation and vacuolar sequestration in roots. In addition, sufficient S application (2 mM) increased the expression of γ-glutamylcysteine synthetase (GSH1) and nicotinamide synthase (NAS) in roots, and excessive S application upregulated the expression of ATP sulfurylase (ATPS) and phytochelatin synthase (PCs). This study provides evidence for the mechanism of mitigating Cd toxicity in pakchoi and will be helpful for developing strategies to reduce Cd accumulation in the edible parts of pakchoi through S fertilizer application.


Assuntos
Brassica/efeitos dos fármacos , Cádmio/metabolismo , Poluentes do Solo/metabolismo , Sulfatos/farmacologia , Aminoaciltransferases/metabolismo , Transporte Biológico , Brassica/crescimento & desenvolvimento , Brassica/metabolismo , Cádmio/toxicidade , Fertilizantes/análise , Glutationa/metabolismo , Hidroponia , Modelos Teóricos , Fitoquelatinas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Poluentes do Solo/toxicidade , Sulfato Adenililtransferase/metabolismo , Sulfatos/metabolismo
16.
ISME J ; 14(2): 450-462, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31659234

RESUMO

Inhibitors can be used to control the functionality of microbial communities by targeting specific metabolisms. The targeted inhibition of dissimilatory sulfate reduction limits the generation of toxic and corrosive hydrogen sulfide across several industrial systems. Sulfate-reducing microorganisms (SRM) are specifically inhibited by sulfate analogs, such as perchlorate. Previously, we showed pure culture SRM adaptation to perchlorate stress through mutation of the sulfate adenylyltransferase, a central enzyme in the sulfate reduction pathway. Here, we explored adaptation to perchlorate across unconstrained SRM on a community scale. We followed natural and bio-augmented sulfidogenic communities through serial transfers in increasing concentrations of perchlorate. Our results demonstrated that perchlorate stress altered community structure by initially selecting for innately more resistant strains. Isolation, whole-genome sequencing, and molecular biology techniques allowed us to define subsequent genetic mechanisms of adaptation that arose across the dominant adapting SRM. Changes in the regulation of divalent anion:sodium symporter family transporters led to increased intracellular sulfate to perchlorate ratios, allowing SRM to escape the effects of competitive inhibition. Thus, in contrast to pure-culture results, SRM in communities cope with perchlorate stress via changes in anion transport and its regulation. This highlights the value of probing evolutionary questions in an ecological framework, bridging the gap between ecology, evolution, genomics, and physiology.


Assuntos
Evolução Molecular , Percloratos/toxicidade , Sulfatos/metabolismo , Ânions/metabolismo , Bactérias/genética , Bactérias/metabolismo , Transporte Biológico , Oxirredução , Percloratos/metabolismo , Sulfato Adenililtransferase/genética
17.
Aquat Toxicol ; 216: 105320, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31590132

RESUMO

Sulfur availability and the end products of its metabolism, cysteine, glutathione and phytochelatins, play an important role in heavy metal tolerance, chromium included. Sulfate and chromate not only compete for the transporters but also for assimilation enzymes and chromium tolerance in various organisms has been associated to differences in this pathway. We investigated the mechanisms of Cr(VI)-tolerance increase induced by S-starvation focusing on the role of ATP sulfurylase (ATS) in two strains of Scenedesmus acutus with different chromium sensitivity. S-starvation enhances the defence potential by increasing sulfate uptake/assimilation and decreasing chromium uptake, thus suggesting a change in the transport system. We isolated two isoforms of the enzyme, SaATS1 and SaATS2, with different sensitivity to sulfur availability, and analysed them in S-sufficient and S-replete condition both in standard and in chromium supplemented medium. SaATS2 expression is different in the two strains and presumably marks a different sulfur perception/exploitation in the Cr-tolerant. Its induction and silencing are compatible with a role in the transient tolerance increase induced by S-starvation. This enzyme can however hardly be responsible for the large cysteine production of the Cr-tolerant strain after starvation, suggesting that cytosolic rather than chloroplastic cysteine production is differently regulated in the two strains.


Assuntos
Cromo/toxicidade , Scenedesmus/metabolismo , Sulfato Adenililtransferase/metabolismo , Enxofre/metabolismo , Biomassa , Cisteína/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Scenedesmus/efeitos dos fármacos , Scenedesmus/enzimologia , Scenedesmus/crescimento & desenvolvimento , Sulfato Adenililtransferase/genética , Fatores de Tempo , Poluentes Químicos da Água/toxicidade
18.
Horm Res Paediatr ; 92(4): 262-268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31461705

RESUMO

OBJECTIVE: Dehydroepiandrosterone (DHEA) sulfotransferase (SULT2A1) converts DHEA to DHEA sulfate (DHEAS) which prevents bioactive androgen excess. This enzymatic reaction requires PAPS (3'-phospho-adenosine-5'-phosphosulfate) biosynthesis mediated by PAPS synthase 2 (PAPSS2). Here, we report a patient presenting with short stature and premature pubarche due to a novel homozygous mutation in the PAPPS2 gene. CASE REPORT: A 7.5-year-old girl was referred for short stature. She was born at term with a birth weight of 2,850 g and her parents were first cousins. At presentation, her height was 113.0 cm (-2.1 SDS) and weight was 28.3 kg (+0.9 SDS), her arm span was 115.0 cm, and upper to lower segment ratio was 1.2. Her pubic hair and breast development were at Tanner stage III and I, respectively. Radiographs revealed mild lumbar scoliosis and platyspondyly and irregular vertebral endplates in the thoracolumbar region. Her serum DHEAS was low (39 ng/mL). The plasma DHEAS/DHEA ratio was significantly decreased on 2 separate measurements (4.4 and 19.8; normal range 31-345). PAPSS2 gene analysis identified a homozygous p.L440Wfs*12 (c.1318_1330 delCTACTACACCCTC) variant. This is the first report of a large deletion leading to a frameshift effect in the PAPSS2 gene and a truncated PAPSS2 protein. CONCLUSION: We describe the third case with PAPSS2 deficiency presenting with premature pubarche, and the first large deletion in the PAPSS2 gene. Although PAPSS2 deficiency is a rare cause of premature pubarche and adrenal androgen excess, it should be considered, especially in cases with disproportionate short stature and clinical hyperandrogenism associated with low plasma DHEAS concentration.


Assuntos
Transtornos do Crescimento/sangue , Complexos Multienzimáticos/genética , Mutação , Puberdade Precoce/sangue , Sulfato Adenililtransferase/genética , Sulfato de Desidroepiandrosterona/sangue , Feminino , Transtornos do Crescimento/genética , Humanos , Puberdade Precoce/genética
19.
Am J Med Genet A ; 179(9): 1884-1894, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31313512

RESUMO

Brachyolmia is a skeletal dysplasia characterized by short spine-short stature, platyspondyly, and minor long bone abnormalities. We describe 18 patients, from different ethnic backgrounds and ages ranging from infancy to 19 years, with the autosomal recessive form, associated with PAPSS2. The main clinical features include disproportionate short stature with short spine associated with variable symptoms of pain, stiffness, and spinal deformity. Eight patients presented prenatally with short femora, whereas later in childhood their short-spine phenotype emerged. We observed the same pattern of changing skeletal proportion in other patients. The radiological findings included platyspondyly, irregular end plates of the elongated vertebral bodies, narrow disc spaces and short over-faced pedicles. In the limbs, there was mild shortening of femoral necks and tibiae in some patients, whereas others had minor epiphyseal or metaphyseal changes. In all patients, exome and Sanger sequencing identified homozygous or compound heterozygous PAPSS2 variants, including c.809G>A, common to white European patients. Bi-parental inheritance was established where possible. Low serum DHEAS, but not overt androgen excess was identified. Our study indicates that autosomal recessive brachyolmia occurs across continents and may be under-recognized in infancy. This condition should be considered in the differential diagnosis of short femora presenting in the second trimester.


Assuntos
Nanismo/genética , Complexos Multienzimáticos/genética , Anormalidades Musculoesqueléticas/genética , Osteocondrodisplasias/genética , Sulfato Adenililtransferase/genética , Adolescente , Adulto , Criança , Pré-Escolar , Nanismo/diagnóstico por imagem , Nanismo/fisiopatologia , Feminino , Genes Recessivos/genética , Predisposição Genética para Doença , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Anormalidades Musculoesqueléticas/diagnóstico por imagem , Anormalidades Musculoesqueléticas/fisiopatologia , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/fisiopatologia , Linhagem , Radiografia , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/fisiopatologia , Adulto Jovem
20.
Int J Phytoremediation ; 21(11): 1112-1121, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044606

RESUMO

Transgenic alfalfa (Medicago sativa L.) plants overexpressing the Arabidopsis ATP sulfurylase gene were generated using Agrobacterium-mediated genetic transformation to enhance their heavy metal accumulation efficiency. The ATP sulfurylase gene was cloned from Arabidopsis, following exposure to vanadium (V) and lead (Pb), and transferred into an Agrobacterium tumefaciens binary vector. This was co-cultivated with leaf explants of the alfalfa genotype Regen SY. Co-cultivated leaf explants were cultured on callus and somatic embryo induction medium, followed by regeneration medium for regenerating complete transgenic plants. The transgenic nature of the plants was confirmed using PCR and southern hybridization. The expression of Arabidopsis ATP sulfurylase gene in the transgenic plants was evaluated through RT-PCR. The selected transgenic lines showed increased tolerance to a mixture of five heavy metals and also demonstrated enhanced metal uptake ability under controlled conditions. The transgenic lines were fertile and did not exhibit any apparent morphological abnormality. The results of this study indicated an effective approach to improve the heavy metal accumulation ability of alfalfa plants which can then be used for the remediation of contaminated soil in arid regions.


Assuntos
Arabidopsis , Metais Pesados , Biodegradação Ambiental , Medicago sativa , Plantas Geneticamente Modificadas , Sulfato Adenililtransferase
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