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1.
PLoS One ; 15(4): e0232220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353014

RESUMO

Terpenes are the largest class of natural products with extensive structural diversity and are widely used as pharmaceuticals, herbicides, flavourings, fragrances, and biofuels. While they have mostly been isolated from plants and fungi, the availability and analysis of bacterial genome sequence data indicates that bacteria also possess many putative terpene synthase genes. In this study, we further explore this potential for terpene synthase activity in bacteria. Twenty two potential class I terpene synthase genes (TSs) were selected to represent the full sequence diversity of bacterial synthase candidates and recombinantly expressed in E. coli. Terpene synthase activity was detected for 15 of these enzymes, and included mono-, sesqui- and diterpene synthase activities. A number of confirmed sesquiterpene synthases also exhibited promiscuous monoterpene synthase activity, suggesting that bacteria are potentially a richer source of monoterpene synthase activity then previously assumed. Several terpenoid products not previously detected in bacteria were identified, including aromandendrene, acora-3,7(14)-diene and longiborneol. Overall, we have identified promiscuous terpene synthases in bacteria and demonstrated that terpene synthases with substrate promiscuity are widely distributed in nature, forming a rich resource for engineering terpene biosynthetic pathways for biotechnology.


Assuntos
Alquil e Aril Transferases/genética , Bactérias/genética , Vias Biossintéticas/genética , Genoma Bacteriano/genética , Filogenia , Terpenos/metabolismo
2.
PLoS Genet ; 16(4): e1008330, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324744

RESUMO

The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria.


Assuntos
Alquil e Aril Transferases/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Alelos , Anticódon , Citosol/enzimologia , Teste de Complementação Genética , Humanos , Mitocôndrias/enzimologia , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Alinhamento de Sequência , Especificidade por Substrato
3.
J Agric Food Chem ; 68(10): 3214-3219, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32079394

RESUMO

Four terpene synthases for the biosynthesis of volatile terpenoids were identified from the transcriptome of Stellera chamaejasme L. flowers, including SchTPS1, SchTPS2, SchTPS3, and SchTPS4. Their functions were characterized by synthetic biology approaches in Escherichia coli and in vitro enzymatic assays. SchTPS1, SchTPS2, and SchTPS3 are guaiene synthases, while SchTPS4 is an (E,E)-geranyl linalool synthase. Next, SchTPS1 and α-guaiene 2-oxidase VvSTO2 were co-expressed in Saccharomyces cerevisiae to reconstruct the biosynthetic pathway of (-)-rotundone, which is a unique aroma compound in fruits, vegetables, and wines. This is the first report for the construction of a (-)-rotundone-producing microbial platform.


Assuntos
Alquil e Aril Transferases/metabolismo , Azulenos/metabolismo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos de Guaiano/metabolismo , Sesquiterpenos/metabolismo , Thymelaeaceae/enzimologia , Alquil e Aril Transferases/genética , Vias Biossintéticas , Flores/enzimologia , Flores/genética , Expressão Gênica , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Thymelaeaceae/genética
4.
Nat Commun ; 11(1): 970, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080200

RESUMO

Deregulation of mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is one of the most common inherited metabolic disorders, due to deficiency of the mitochondrial methylmalonyl-coenzyme A mutase (MMUT). How MMUT deficiency triggers cell damage remains unknown, preventing the development of disease-modifying therapies. Here we combine genetic and pharmacological approaches to demonstrate that MMUT deficiency induces metabolic and mitochondrial alterations that are exacerbated by anomalies in PINK1/Parkin-mediated mitophagy, causing the accumulation of dysfunctional mitochondria that trigger epithelial stress and ultimately cell damage. Using drug-disease network perturbation modelling, we predict targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient-derived cells and alleviate phenotype changes in mmut-deficient zebrafish. These results suggest a link between primary MMUT deficiency, diseased mitochondria, mitophagy dysfunction and epithelial stress, and provide potential therapeutic perspectives for MMA.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Metilmalonil-CoA Mutase/deficiência , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Mitofagia/fisiologia , Alquil e Aril Transferases/deficiência , Alquil e Aril Transferases/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Erros Inatos do Metabolismo/genética , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Camundongos Knockout , Doenças Mitocondriais/genética , Mitofagia/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estresse Fisiológico , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Peixe-Zebra
5.
Plant Mol Biol ; 102(4-5): 517-535, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927660

RESUMO

KEY MESSAGE: We reported the functional characterization of cDNAs encoding short-chain isoprenyl diphosphate synthases that control the partitioning of precursors for lavender terpenoids. Lavender essential oil is composed of regular and irregular monoterpenes, which are derived from linear precursors geranyl diphosphate (GPP) and lavandulyl diphosphate (LPP), respectively. Although this plant strongly expresses genes responsible for the biosynthesis of both monoterpene classes, it is unclear why regular monoterpenes dominate the oil. Here, we cloned and characterized Lavandula x intermedia cDNAs encoding geranyl diphosphate synthase (LiGPPS), geranylgeranyl diphosphate synthase (LiGGPPS) and farnesyl diphosphate synthase (LiFPPS). LiGPPS was heteromeric protein, consisting of a large subunit (LiGPPS.LSU) and a small subunit for which two different cDNAs (LiGPPS.SSU1 and LiGPPS.SSU2) were detected. Neither recombinant LiGPPS subunits was active by itself. However, when co-expressed in E. coli LiGPPS.LSU and LiGPPS.SSU1 formed an active heteromeric GPPS, while LiGPPS.LSU and LiGPPS.SSU2 did not form an active protein. Recombinant LiGGPPS, LiFPPS and LPP synthase (LPPS) proteins were active individually. Further, LiGPPS.SSU1 modified the activity of LiGGPPS (to produce GPP) in bacterial cells co-expressing both proteins. Given this, and previous evidence indicating that GPPS.SSU can modify the activity of GGPPS to GPPS in vitro and in plants, we hypothesized that LiGPPS.SSU1 modifies the activity of L. x intermedia LPP synthase (LiLPPS), thus accounting for the relatively low abundance of LPP-derived irregular monoterpenes in this plant. However, LiGPPS.SSU1 did not affect the activity of LiLPPS. These results, coupled to the observation that LiLPPS transcripts are more abundant than those of GPPS subunits in L. x intermedia flowers, suggest that regulatory mechanisms other than transcriptional control of LPPS regulate precursor partitioning in lavender flowers.


Assuntos
Alquil e Aril Transferases/metabolismo , Dimetilaliltranstransferase/metabolismo , Flores/enzimologia , Lavandula/enzimologia , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/genética , Catálise , Dimetilaliltranstransferase/genética , Difosfatos , Diterpenos , Escherichia coli/genética , Escherichia coli/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Lavandula/genética , Monoterpenos/metabolismo , Fases de Leitura Aberta , Fenótipo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Terpenos/metabolismo , Tabaco/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(3): 1711-1721, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31811023

RESUMO

Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent-response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance.


Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Alquil e Aril Transferases/genética , Antifúngicos/farmacologia , Aspergillus fumigatus/genética , Quelantes de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Quitina Sintase/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Heme/análogos & derivados , Heme/biossíntese , Humanos , Proteínas de Membrana/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Fosfoproteínas Fosfatases/metabolismo , Espécies Reativas de Oxigênio , Fatores de Transcrição/metabolismo , Triazóis/farmacologia
7.
Phytochemistry ; 169: 112126, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31644985

RESUMO

Thyme (Thymus spp.) is a valuable genus of Lamiaceae family with different pharmaceutical and food properties. Thymol has also been considered as the major essential oil compound in most of the studied Thymus species. In this research, the gene encoding γ-terpinene synthase (Ttps2) was sequenced in T. vulgaris and in eight Iranian thymes including T. carmanicus, T. daenensis, T. fedtschenkoi, T. kotschyanus, T. migricus, T. pubescens, T. serpyllum, and T. trautvetteri. Genetic relationships based on terpene synthase genes were also determined among the studied species. Rapid Amplification of cDNA Ends (RACE) PCR was done to complete the sequence of all species. The cDNA of the studied species possessed an open reading frame ranging from 1788 to 1794 bp that encode for a protein of 596-598 amino acids, presenting all the conserved motifs characteristics of monoterpene synthases. The taxonomic status of Thymus species was determined based on eight reported sections. The species were classified in three major groups. The first and second group comprised species of Micantes and Mastichina sections. The third cluster included the species belonging to Serpyllum and Pseudothymbra sections. Overall, phylogenetic analysis according to whole sequence of Ttps2 gene can help providing insights in respect to its evolutionary process. Finally, clustering based on the amount of main essential oils components (thymol and carvacrol) was compared with that based on Ttps2 gene classification in the studied Thymus species, showing that clustering is not always in accordance.


Assuntos
Alquil e Aril Transferases/genética , Timol/metabolismo , Thymus (Planta)/química , Alquil e Aril Transferases/metabolismo , Variação Genética/genética , Estrutura Molecular , Análise de Sequência de Proteína , Timol/química
8.
Nucleic Acids Res ; 48(3): 1435-1450, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31863583

RESUMO

tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.


Assuntos
Alquil e Aril Transferases/genética , Proteínas de Bactérias/genética , RNA de Transferência/genética , Alquil e Aril Transferases/química , Proteínas de Bactérias/química , Escherichia coli/enzimologia , Escherichia coli/genética , Conformação de Ácido Nucleico , Nucleotídeos , Saccharomyces cerevisiae/enzimologia
9.
J Agric Food Chem ; 68(2): 678-685, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31858793

RESUMO

Elaeis guineensis is a tropical oil crop and has the highest oil yield per unit area. Palm oil has high palmitic acid content and is also rich in vitamins, including vitamin E. We conducted genome-wide association studies in a diversity panel of 161 E. guineensis accessions to identify single-nucleotide polymorphisms (SNPs) linked with vitamin E and validated candidate genes in these marker-associated intervals. Based on the SNPs reported in our previous research, 47 SNP markers were detected to be significantly associated with the variation of tocopherol and tocotrienol content at a cutoff P value of 6.3 × 10-7. A total of 656 candidate genes in the flanking regions of the 47 SNPs were identified, followed by pathway enrichment analysis. Of these candidate genes, EgHGGT (homogentisate geranylgeranyl transferase) involved in the biosynthesis of tocotrienols had a higher expression level in the mesocarp compared to other tissues. Expression of the EgHGGT gene was positively correlated with the variation in α-tocotrienol content. Induced overexpression of the gene in Arabidopsis caused a significant increase in vitamin E content and production of α-tocotrienols compared to wild Arabidopsis.


Assuntos
Arecaceae/metabolismo , Genoma de Planta , Vitamina E/biossíntese , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Arecaceae/enzimologia , Arecaceae/genética , Vias Biossintéticas , Estudo de Associação Genômica Ampla , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único
10.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(12): 1158-1162, 2019 Dec 10.
Artigo em Chinês | MEDLINE | ID: mdl-31813137

RESUMO

OBJECTIVE: To report on clinical characteristics and genetic findings in 15 Chinese patients with methylmalonic acidemia (MMA). METHODS: For the 15 MMA patients detected by tandem mass spectrometry, genetic analysis was carried out in twelve pedigrees. Clinical characteristics, genetic finding, treatment and outcomes were retrospectively analyzed. RESULTS: The main features of the patients included poor feeding, recurrent vomiting, lethargy, seizure and development retardation. Blood propionylcarnitine (except for 3 patients), its ratio with acetylcarnitine, and urine methylmalonic acid were increased in all patients. Twelve patients were diagnosed genetically, which included 7 with MUT variants, 4 with MMACHC variants, and 1 with MMAB variant. Nine MUT variants were detected, among which c.1159A>C, 753+1delGinsTGGTTATTA and c.504del were novel. Six known pathogenic MMACHC variants and two novel MMAB variants (c.289_290delGG, c.566G>A) were also detected. Seven patients died of metabolic crises within a year, others had improved effectively following the treatment, but had mild to severe growth delay and/or developmental retardation. CONCLUSION: The clinical manifestation of MMA are complex. Most patients have variants of the MUT and MMACHC genes. High mortality may occur before one year of age.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Alquil e Aril Transferases/genética , China , Humanos , Metilmalonil-CoA Mutase/genética , Oxirredutases/genética , Linhagem , Estudos Retrospectivos
11.
Genes (Basel) ; 10(12)2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783675

RESUMO

Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Neoplasias Pulmonares/mortalidade , Fator de Transcrição Sp1/genética , Células A549 , Alquil e Aril Transferases/química , Alquil e Aril Transferases/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Análise de Sobrevida , Sítio de Iniciação de Transcrição
12.
ACS Chem Biol ; 14(12): 2922-2931, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31756078

RESUMO

Dimethylallyltryptophan synthases catalyze the regiospecific transfer of (oligo)prenylpyrophosphates to aromatic substrates like tryptophan derivatives. These reactions are key steps in many biosynthetic pathways of fungal and bacterial secondary metabolites. In vitro investigations on recombinant DMATS1Ff from Fusarium fujikuroi identified the enzyme as the first selective reverse tryptophan-N-1 prenyltransferase of fungal origin. The enzyme was also able to catalyze the reverse N-geranylation of tryptophan. DMATS1Ff was shown to be phylogenetically related to fungal tyrosine O-prenyltransferases and fungal 7-DMATS. Like these enzymes, DMATS1Ff was able to convert tyrosine into its regularly O-prenylated derivative. Investigation of the binding sites of DMATS1Ff by homology modeling and comparison to the crystal structure of 4-DMATS FgaPT2 showed an almost identical site for DMAPP binding but different residues for tryptophan coordination. Several putative active site residues were verified by site directed mutagenesis of DMATS1Ff. Homology models of the phylogenetically related enzymes showed similar tryptophan binding residues, pointing to a unified substrate binding orientation of tryptophan and DMAPP, which is distinct from that in FgaPT2. Isotopic labeling experiments showed the reaction catalyzed by DMATS1Ff to be nonstereospecific. Based on these data, a detailed mechanism for DMATS1Ff catalysis is proposed.


Assuntos
Alquil e Aril Transferases/metabolismo , Fusarium/enzimologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Sítios de Ligação , Domínio Catalítico , Mutagênese Sítio-Dirigida , Conformação Proteica
13.
BMC Med Genet ; 20(1): 165, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660881

RESUMO

BACKGROUND: Treatment of steroid-resistant nephrotic syndrome (SRNS) remains a challenge for paediatricians. SRNS accounts for 10~20% of childhood cases of nephrotic syndrome (NS). Individuals with SRNS overwhelmingly progress to chronic kidney disease (CKD) and end-stage kidney disease (ESRD). Genetic research is of great significance for diagnosis and treatment. More than 39 recessive or dominant genes have been found to cause human SRNS, including COQ2. COQ2 gene mutations not only cause primary coenzyme Q10 deficiency but also cause SRNS without extrarenal manifestations. The concept of COQ2 nephropathy has been proposed for a long time. Mutations in the COQ2 gene have rarely been reported. Worldwide, only 5 cases involving 4 families have been reported. CASE PRESENTATION: We present the case of a 6-month-old girl with steroid-resistant glomerulopathy due to a COQ2 defect with no additional systemic symptoms. The patient was identified as a homozygote for the c.832 T > C (p. Cys278Arg) missense mutation and a single base homozygous mutation in ARSB gene in c.1213 + 1G > A. The father and mother were heterozygous mutation carriers in both COQ2 and ARSB, and her healthy sister was only a heterozygous mutation carrier in COQ2. In this case, hormone therapy was ineffective, and progressive deterioration of renal function occurred within 1 week after onset, leading to acute renal failure and eventual death. CONCLUSIONS: We reported a consanguinity married family which had COQ2 and ARSB dual mutant. Kidney diseases caused by COQ2 gene mutations can manifest as SRNS, with poor prognosis. The C. 832 T > c (p.csc 278arg) is a new mutation site. Genetic assessment for children with steroid-resistant nephrotic syndrome, especially in infancy, is very important. Families with a clear family history should receive genetic counselling and prenatal examinations, and children without a family phenotype should also receive genetic screening as early as possible.


Assuntos
Alquil e Aril Transferases/genética , Consanguinidade , Casamento , Metilprednisolona/uso terapêutico , Mutação , N-Acetilgalactosamina-4-Sulfatase/genética , Síndrome Nefrótica/genética , Resistência a Medicamentos , Evolução Fatal , Feminino , Triagem de Portadores Genéticos , Homozigoto , Humanos , Lactente , Masculino , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/terapia , Linhagem , Diálise Peritoneal
14.
Plant Sci ; 289: 110277, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31623780

RESUMO

Along with the species evolution, plants have evolved ways to produce a different collection of terpenoids to accommodate its biotic and abiotic environment, and terpene synthase (TPS) is one of the major contributors to various terpene compounds. The timber of a monotypic and relictual conifer species of Cupressace, Taiwania cryptomerioides, has excellent durability, and one of the essential factors for Taiwania to resist decay and insect pests is sesquiterpene. Compared to other conifers, Taiwania has much higher abundance of cadinene-type sesquiterpenes, and the presence of cedrene-type sesquiterpenes. To understand sesquiterpene biosynthesis in Taiwania, we functionally characterized 10 T. cryptomerioides TPSs (TcTPSs) in vivo or in planta, which could catalyze sesquiterpene formation and potentially are involved in biosynthesis of diverse sesquiterpenoids in Taiwania. The distant phylogenetic relationship and the intron loss event of TcTPSs correlate to the differentiation of chemical profile Taiwania compared to other conifers. Furthermore, we identified TcTPS3 and TcTPS12 as δ-cadinene synthase, and TcTPS6 as cedrol synthase, which demonstrates the important contributions of dynamic evolution in TPSs to the chemical diversity in plants. Combining with functional characterization and comparison of catalytic residues, we conclude at least three catalytic routes for sesquiterpene biosynthesis in this species, and the skeleton diversity has been expended in T. cryptomeriodes.


Assuntos
Alquil e Aril Transferases/genética , Cupressaceae/genética , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo , Transcriptoma , Alquil e Aril Transferases/metabolismo , Cupressaceae/metabolismo , Filogenia , Proteínas de Plantas/metabolismo
15.
Nat Commun ; 10(1): 3975, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484924

RESUMO

Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1, but not Rhoa and Cdc42, reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.


Assuntos
Imunidade Inata/genética , Prenilação de Proteína , Transdução de Sinais/genética , Proteínas rac1 de Ligação ao GTP/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Citocinas/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica , Células RAW 264.7 , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
16.
Appl Microbiol Biotechnol ; 103(21-22): 8785-8797, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31515597

RESUMO

Ophiobolins (ophs) are characteristic 5-8-5 tricyclic sesterterpenes with potential pharmaceutical activities. Ophiobolin synthase is a bifunctional terpene synthase (BTS) that catalyzes both chain elongation and cyclization. In Aspergillus ustus 094102, ophiobolin accumulation was involved with not only ophiobolin synthase C25 (Au8003) but also other four gene clusters containing C15 (Au6298), C20 (Au13192 and Au11565), and C30 (Au3446) terpene synthases. In this report, overexpression of codon-optimized gene Au8003 resulted in a detectable production of oph F in E. coli. In subsequent modulation of culture conditions, pentose arabinose allowed a more than 10-fold improvement of production than that of glycerol. To achieve a higher titer, the whole mevalonate pathway and an additional copy of isopentenyl diphosphate isomerase gene were assembled, leading to approximately 24-fold and 60-fold yield increases, respectively. The above four terpene synthase genes related to ophiobolin production in strain 094102 were individually or combinatorially overexpressed with Au8003 to mimic the original fungal biosynthesis. The biosynthesis of oph scaffold was increased by short-chain terpene synthases (C15 and C20), among which the C15 synthase gene contributed the highest yield of 82.76 mg/L at 96 h; the multi-gene combinatorial results suggested that cyclization might be a rate-limiting step. Further protein engineering including fusion tags and phylogenetically based mutations on the rate-limiting cyclization part of Au8003 enabled a further yield improvement (> 150 mg/L at 96 h) in shake flasks. These multiple approaches for sesterterpene skeleton production using engineered E. coli may be applicable for cost-effective, high-yield productions of ophiobolins and other compounds synthesized by BTSs.


Assuntos
Alquil e Aril Transferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Sesterterpenos/metabolismo , Alquil e Aril Transferases/genética , Aspergillus/enzimologia , Proteínas Fúngicas/genética , Engenharia Metabólica , Família Multigênica
17.
PLoS One ; 14(9): e0222363, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31513654

RESUMO

Terpenes are responsible for most or all of the odor and flavor properties of Cannabis sativa, and may also impact effects users experience either directly or indirectly. We report the diversity of terpene profiles across samples bound for the Washington dispensary market. The remarkable degree of variation in terpene profiles ultimately results from action of a family of terpene synthase genes, only some of which have been described. Using a recently available genome assembly we describe 55 terpene synthases with genomic context, and tissue specific expression. The family is quite diverse from a protein similarity perspective, and subsets of the family are expressed in all tissues in the plant, including a set of root specific monoterpene synthases that could well have agronomic importance. Ultimately understanding and breeding for specific terpene profiles will require a good understanding of the gene family that underlies it. We intend for this work to serve as a foundation for that.


Assuntos
Alquil e Aril Transferases/genética , Cannabis/genética , Terpenos/metabolismo , Alquil e Aril Transferases/metabolismo , Cannabis/química , Clonagem Molecular/métodos , Evolução Molecular , Flores/genética , Genes de Plantas , Genoma de Planta/genética , Genômica , Filogenia , Terpenos/química
18.
Plant Sci ; 287: 110187, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481200

RESUMO

Almond (Prunus dulcis) is an agricultural and economically important fruit tree from the Rosaceae family used in the food industry. The monoterpenes and sesquiterpenes perform important ecological functions such as insecticidal and antifeedant activities against various insects. The young fruits of the different almond varieties were found to produce considerable amounts of terpene volatiles, including linalool and geraniol. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, existing genome databases of the Rosaceae were screened for almond genes with significant sequence similarity to other plants TPSs. Bioinformatics analysis led to the identification of seven putative TPSs genes with complete open reading frames. We characterized the enzymes encoded by these seven complementary DNAs: the monoterpene synthases PdTPS1, PdTPS3, PdTPS5, and PdTPS6 belong to the TPS-b clade, which catalyzes the formation of ß-phellandrene, geraniol, linalool, and farnesene, respectively. The sesquiterpene synthases PdTPS2 and PdTPS4, which belong to the TPS-a clade mainly catalyze the formation of bergamotene, while another sesquiterpene synthase, PdTPS7, from the TPS-g clade showed nerolidol synthase activity. The qRT-PCR analysis revealed that the various tissues of almond varieties showed differential transcription for all these PdTPSs genes.


Assuntos
Alquil e Aril Transferases/metabolismo , Prunus dulcis/enzimologia , Terpenos/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Monoterpenos Acíclicos/metabolismo , Alquil e Aril Transferases/genética , Biologia Computacional , Monoterpenos Cicloexânicos/metabolismo , Frutas/enzimologia , Frutas/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prunus dulcis/genética
19.
Biochem J ; 476(18): 2607-2621, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31484677

RESUMO

All land plants contain at least one class II diterpene cyclase (DTC), which utilize an acid-base catalytic mechanism, for the requisite production of ent-copalyl diphosphate (ent-CPP) in gibberellin A (GA) phytohormone biosynthesis. These ent-CPP synthases (CPSs) are hypothesized to be derived from ancient bacterial origins and, in turn, to have given rise to the frequently observed additional DTCs utilized in more specialized plant metabolism. However, such gene duplication and neo-functionalization has occurred repeatedly, reducing the utility of phylogenetic analyses. Support for evolutionary scenarios can be found in more specific conservation of key enzymatic features. While DTCs generally utilize a DxDD motif as the catalytic acid, the identity of the catalytic base seems to vary depending, at least in part, on product outcome. The CPS from Arabidopsis thaliana has been found to utilize a histidine-asparagine dyad to ligate a water molecule that serves as the catalytic base, with alanine substitution leading to the production of 8ß-hydroxy-ent-CPP. Here this dyad and effect of Ala substitution is shown to be specifically conserved in plant CPSs involved in GA biosynthesis, providing insight into plant DTC evolution and assisting functional assignment. Even more strikingly, while GA biosynthesis arose independently in plant-associated bacteria and fungi, the catalytic base dyad also is specifically found in the relevant bacterial, but not fungal, CPSs. This suggests functional conservation of CPSs from bacteria to plants, presumably reflecting an early role for derived diterpenoids in both plant development and plant-microbe interactions, eventually leading to GA, and a speculative evolutionary scenario is presented.


Assuntos
Alquil e Aril Transferases , Proteínas de Arabidopsis , Arabidopsis , Bactérias , Proteínas de Bactérias , Evolução Molecular , Giberelinas/metabolismo , Filogenia , Proteínas de Plantas , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
20.
Planta ; 250(5): 1687-1702, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31414203

RESUMO

MAIN CONCLUSION: The study performed genome-wide identification, characterization and evolution analysis of gene clusters for phytoalexin terpenoid biosynthesis in tobacco, and specifically illustrated ones for capsidiol, an efficient defensive specialized metabolite. Terpenoid phytoalexins play an important role in plant self-defense against pest and pathogen attack. Terpenoid biosynthesis involves terpene synthase and cytochrome P450, which always locate and function as cluster(s). In this study, we performed genome-wide investigation of metabolic gene clusters involved in terpenoid production in tobacco (Nicotiana tabacum). Due to the complexity of the tobacco genome, we modified a published prediction pipeline to reduce the influence of the large number of repeats and to improve the annotation of tobacco genes with respect to their metabolic functions. We identified 1181 metabolic gene clusters with 34 of them potentially being involved in terpenoid biosynthesis. Through integration with transcriptome and metabolic pathway annotation analyses, 3 of the 34 terpenoid biosynthesis-related gene clusters were determined to be high-confidence ones, with 2 involved in biosynthesis of capsidiol, a terpenoid recognized as 1 of the effective resistance compounds in the Nicotiana species. The capsidiol-related gene cluster was conserved in N. sylvestris, N. tomentosiformis and N. attenuate. Our findings demonstrate that phytoalexins in tobacco can arise from operon-like gene clusters, a genomic pattern characterized as being beneficial for rapid stress response, gene co-regulation, co-function and co-heredity.


Assuntos
Alquil e Aril Transferases/metabolismo , Regulação da Expressão Gênica de Plantas , Sesquiterpenos/metabolismo , Terpenos/metabolismo , Tabaco/genética , Transcriptoma , Alquil e Aril Transferases/genética , Família Multigênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/metabolismo
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