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1.
Int J Mol Sci ; 22(15)2021 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-34360673

RESUMO

Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.


Assuntos
Arabidopsis/enzimologia , Sementes/enzimologia , Transdução de Sinais , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Mutação , Tubo Polínico/fisiologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
2.
Org Biomol Chem ; 19(30): 6650-6656, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34264250

RESUMO

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Biocatálise , Alquil e Aril Transferases/genética , Domínio Catalítico , Ciclização , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Tabaco/enzimologia
3.
Biomolecules ; 11(6)2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208762

RESUMO

Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica, a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene (SiTPS). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS-over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization.


Assuntos
Alquil e Aril Transferases/isolamento & purificação , Basidiomycota/enzimologia , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Basidiomycota/metabolismo , Endófitos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Lycopersicon esculentum/metabolismo , Raízes de Plantas/metabolismo , Simbiose/genética , Terpenos/química , Terpenos/metabolismo
4.
Int J Biol Macromol ; 185: 949-958, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34237366

RESUMO

Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii, we observed that Ses-TwTPS1-1 and Ses-TwTPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses-TwTPS1-2, Ses-TwTPS3, and Ses-TwTPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E, E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses-TwTPS1-1:V199I and Ses-TwTPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Tripterygium/enzimologia , Alquil e Aril Transferases/química , Domínio Catalítico , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Terpenos/metabolismo , Tripterygium/genética
5.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203299

RESUMO

Terpenoids are the largest class of plant secondary metabolites and are one of the major emitted volatile compounds released to the atmosphere. They have functions of attracting pollinators or defense function, insecticidal properties, and are even used as pharmaceutical agents. Because of the importance of terpenoids, an increasing number of plants are required to investigate the function and evolution of terpene synthases (TPSs) that are the key enzymes in terpenoids biosynthesis. Orchidacea, containing more than 800 genera and 28,000 species, is one of the largest and most diverse families of flowering plants, and is widely distributed. Here, the diversification of the TPSs evolution in Orchidaceae is revealed. A characterization and phylogeny of TPSs from four different species with whole genome sequences is available. Phylogenetic analysis of orchid TPSs indicates these genes are divided into TPS-a, -b, -e/f, and g subfamilies, and their duplicated copies are increased in derived orchid species compared to that in the early divergence orchid, A. shenzhenica. The large increase of both TPS-a and TPS-b copies can probably be attributed to the pro-duction of different volatile compounds for attracting pollinators or generating chemical defenses in derived orchid lineages; while the duplications of TPS-g and TPS-e/f copies occurred in a species-dependent manner.


Assuntos
Alquil e Aril Transferases/metabolismo , Orchidaceae/enzimologia , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/genética , Evolução Molecular , Filogenia , Proteínas de Plantas/genética
6.
BMC Plant Biol ; 21(1): 354, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315414

RESUMO

BACKGROUND: Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood. RESULTS: In this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson's correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. CONCLUSION: The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.


Assuntos
Atractylodes/metabolismo , Perfilação da Expressão Gênica/métodos , Transcriptoma/genética , Alquil e Aril Transferases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Transferases Intramoleculares/metabolismo , Análise de Sequência de RNA/métodos , Sesquiterpenos/metabolismo , Esqualeno Mono-Oxigenase/metabolismo
7.
Plant Sci ; 309: 110952, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34134848

RESUMO

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool.


Assuntos
Acetatos/metabolismo , Monoterpenos Acíclicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclopentanos/metabolismo , Dendrobium/genética , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Vias Biossintéticas , Dendrobium/química , Dendrobium/metabolismo , Flores/química , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Óleos Voláteis/metabolismo , Óleos Vegetais/metabolismo , Proteínas de Plantas/genética
8.
Nat Commun ; 12(1): 3487, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108468

RESUMO

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.


Assuntos
Alquil e Aril Transferases/química , Diterpenos/metabolismo , Proteínas Fúngicas/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Ciclização , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeos/biossíntese , Liases/química , Liases/metabolismo , Enzimas Multifuncionais , Fosfatos de Poli-Isoprenil/metabolismo , Conformação Proteica
9.
Plant Cell Rep ; 40(7): 1269-1284, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34052884

RESUMO

KEY MESSAGE: Herein, 37 ARF genes were identified and analyzed in Hedychium coronarium and HcARF5 showed a potential role in the regulation of HcTPS3. Auxin is an important plant hormone, implicated in various aspects of plant growth and development processes especially in the biosynthesis of various secondary metabolites. Auxin response factors (ARF) belong to the transcription factors (TFs) gene family and play a crucial role in transcriptional activation/repression of auxin-responsive genes by directly binding to their promoter region. Nevertheless, whether ARF genes are involved in the regulatory mechanism of volatile compounds in flowering plants is largely unknown. ß-ocimene is a key floral volatile compound synthesized by terpene synthase 3 (HcTPS3) in Hedychium coronarium. A comprehensive analysis of H. coronarium genome reveals 37 candidate ARF genes in the whole genome. Tissue-specific expression patterns of HcARFs family members were assessed using available transcriptome data. Among them, HcARF5 showed a higher expression level in flowers, and significantly correlated with the key structural ß-ocimene synthesis gene (HcTPS3). Furthermore, transcript levels of both genes were associated with the flower development. Under hormone treatments, the response of HcARF5 and HcTPS3, and the emission level of ß-ocimene contents were evaluated. Subcellular and transcriptional activity assay showed that HcARF5 localizes to the nucleus and possesses transcriptional activity. Yeast one-hybrid (Y1H) and dual-luciferase assays revealed that HcARF5 directly regulates the transcriptional activity of HcTPS3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that HcARF5 interacts with scent-related HcIAA4, HcIAA6, and HcMYB1 in vivo. Overall, these results indicate that HcARF5 is potentially involved in the regulation of ß-ocimene synthesis in H. coronarium.


Assuntos
Monoterpenos Acíclicos/metabolismo , Alcenos/metabolismo , Alquil e Aril Transferases/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Zingiberaceae/genética , Alquil e Aril Transferases/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , MicroRNAs , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Zingiberaceae/efeitos dos fármacos , Zingiberaceae/metabolismo
10.
Chem Pharm Bull (Tokyo) ; 69(5): 444-446, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33952854

RESUMO

Structurally diverse fungal meroterpenoids are promising drug seed compounds. To obtain unnatural, novel meroterpene scaffolds, we tested combinatorial biosynthesis by co-expressing functionally distinct terpene cyclase (TPC) genes, pyr4, ascF, andB, or cdmG, with the biosynthetic genes for the production of a TPC substrate, (10'R)-epoxyfarnesyl-dimethylorsellinic acid-3,5-methyl ester, in Aspergillus oryzae NSAR1 as a heterologous host. As a result, all of the tested TPCs afforded the same two novel mono-cyclization products. This study provides important information on the substrate scope of the TPCs, and will contribute to the production of unnatural, novel molecules for future drug discovery.


Assuntos
Alquil e Aril Transferases/metabolismo , Ciclopentanos/metabolismo , Terpenos/metabolismo , Alquil e Aril Transferases/genética , Aspergillus oryzae/enzimologia , Biocatálise , Ciclopentanos/química , Estrutura Molecular , Terpenos/química
11.
Nat Immunol ; 22(7): 904-913, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34031613

RESUMO

Antigen-activated B cells diversify variable regions of B cell antigen receptors by somatic hypermutation in germinal centers (GCs). The positive selection of GC B cells that acquire high-affinity mutations enables antibody affinity maturation. In spite of considerable progress, the genomic states underlying this process remain to be elucidated. Single-cell RNA sequencing and topic modeling revealed increased expression of the oxidative phosphorylation (OXPHOS) module in GC B cells undergoing mitoses. Coupled analysis of somatic hypermutation in immunoglobulin heavy chain (Igh) variable gene regions showed that GC B cells acquiring higher-affinity mutations had further elevated expression of OXPHOS genes. Deletion of mitochondrial Cox10 in GC B cells resulted in reduced cell division and impaired positive selection. Correspondingly, augmentation of OXPHOS activity with oltipraz promoted affinity maturation. We propose that elevated OXPHOS activity promotes B cell clonal expansion and also positive selection by tuning cell division times.


Assuntos
Linfócitos B/metabolismo , Perfilação da Expressão Gênica , Centro Germinativo/metabolismo , Mutação , Fosforilação Oxidativa , Receptores de Antígenos de Linfócitos B/genética , Análise de Célula Única , Transcriptoma , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Linfócitos B/imunologia , Proliferação de Células , Células Cultivadas , Análise Mutacional de DNA , Feminino , Genes de Cadeia Pesada de Imunoglobulina , Centro Germinativo/imunologia , Região Variável de Imunoglobulina , Ativação Linfocitária , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA-Seq , Receptores de Antígenos de Linfócitos B/metabolismo
12.
Genes (Basel) ; 12(4)2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33918244

RESUMO

Terpenoids are a wide variety of natural products and terpene synthase (TPS) plays a key role in the biosynthesis of terpenoids. Mentha plants are rich in essential oils, whose main components are terpenoids, and their biosynthetic pathways have been basically elucidated. However, there is a lack of systematic identification and study of TPS in Mentha plants. In this work, we genome-widely identified and analyzed the TPS gene family in Mentha longifolia, a model plant for functional genomic research in the genus Mentha. A total of 63 TPS genes were identified in the M. longifolia genome sequence assembly, which could be divided into six subfamilies. The TPS-b subfamily had the largest number of genes, which might be related to the abundant monoterpenoids in Mentha plants. The TPS-e subfamily had 18 members and showed a significant species-specific expansion compared with other sequenced Lamiaceae plant species. The 63 TPS genes could be mapped to nine scaffolds of the M. longifolia genome sequence assembly and the distribution of these genes is uneven. Tandem duplicates and fragment duplicates contributed greatly to the increase in the number of TPS genes in M. longifolia. The conserved motifs (RR(X)8W, NSE/DTE, RXR, and DDXXD) were analyzed in M. longifolia TPSs, and significant differentiation was found between different subfamilies. Adaptive evolution analysis showed that M. longifolia TPSs were subjected to purifying selection after the species-specific expansion, and some amino acid residues under positive selection were identified. Furthermore, we also cloned and analyzed the catalytic activity of a single terpene synthase, MlongTPS29, which belongs to the TPS-b subfamily. MlongTPS29 could encode a limonene synthase and catalyze the biosynthesis of limonene, an important precursor of essential oils from the genus Mentha. This study provides useful information for the biosynthesis of terpenoids in the genus Mentha.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Mentha/enzimologia , Análise de Sequência de DNA/métodos , Adaptação Biológica , Mapeamento Cromossômico/métodos , Evolução Molecular , Duplicação Gênica , Genoma de Planta , Limoneno/metabolismo , Mentha/genética , Mentha/metabolismo , Família Multigênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Seleção Genética
13.
Molecules ; 26(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801907

RESUMO

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.


Assuntos
Alquil e Aril Transferases/genética , Confrei/genética , Alcaloides de Pirrolizidina/metabolismo , Alquil e Aril Transferases/metabolismo , Boraginaceae/genética , Boraginaceae/metabolismo , Sistemas CRISPR-Cas/genética , Confrei/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica de Plantas/genética , Raízes de Plantas/genética , Plantas Medicinais/genética , Alcaloides de Pirrolizidina/química
14.
Int J Mol Sci ; 22(5)2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33801503

RESUMO

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.


Assuntos
Alquil e Aril Transferases/metabolismo , Inibidores Enzimáticos/farmacologia , Fragmentos de Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas rap de Ligação ao GTP/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Humanos , Prenilação de Proteína , Especificidade por Substrato , Xantinas/farmacologia , Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/genética
15.
PLoS Comput Biol ; 17(3): e1008197, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33750949

RESUMO

Sesquiterpene synthases (STSs) catalyze the formation of a large class of plant volatiles called sesquiterpenes. While thousands of putative STS sequences from diverse plant species are available, only a small number of them have been functionally characterized. Sequence identity-based screening for desired enzymes, often used in biotechnological applications, is difficult to apply here as STS sequence similarity is strongly affected by species. This calls for more sophisticated computational methods for functionality prediction. We investigate the specificity of precursor cation formation in these elusive enzymes. By inspecting multi-product STSs, we demonstrate that STSs have a strong selectivity towards one precursor cation. We use a machine learning approach combining sequence and structure information to accurately predict precursor cation specificity for STSs across all plant species. We combine this with a co-evolutionary analysis on the wealth of uncharacterized putative STS sequences, to pinpoint residues and distant functional contacts influencing cation formation and reaction pathway selection. These structural factors can be used to predict and engineer enzymes with specific functions, as we demonstrate by predicting and characterizing two novel STSs from Citrus bergamia.


Assuntos
Alquil e Aril Transferases/metabolismo , Evolução Molecular , Aprendizado de Máquina , Plantas/enzimologia , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/química , Sequência de Aminoácidos , Cátions , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
16.
Plant Sci ; 304: 110790, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568294

RESUMO

Chamaecyparis formosensis Matsum. is an endemic and precious coniferous species of Taiwan, and is known for a high abundance of specialized metabolites, which contributes to the excellent timber durability. Several terpenoids were identified and isolated from C. formosensis wood and needles, and exhibit anti-fungal and anti-bacterial bioactivities, which may participate in plant defense against pathogens. In various identified compounds, not only cadinene and ferruginol, were identified in C. formosensis extracts but also unique diterpenoids, which include pisferal, totarol, and derivates of isoabienol. To understand the biosynthesis of these specific diterpenoids, we conducted a series of functional characterization of the C. formosensis diterpene synthases (CfdiTPSs), which participate in skeleton formation and differentiation of diterpenes. In this study, we identified eight diTPSs from C. formosensis transcriptome, and they all contain either class I or class II motif, which indicates they are all monofunctional enzymes. These candidates consist of three class II diTPSs and five class I diTPSs, and after conducting in vivo and in vitro assays, class II diTPS CfCPS1 was characterized as a (+)-copalyl diphosphate synthase ((+)-CPS), and class I diTPSs CfKSL1 could further convert (+)-copalyl diphosphate ((+)-CPP) to levopimaradiene. Meanwhile, CfKSL1 also accepted labda-13-en-8-ol diphosphate (LPP) as substrate and formed monoyl oxide. Another class I diTPS, CfKSL4, exhibits a strong enzymatic ability of isoabienol synthase, which is firstly reported in conifer. This finding provides potential participants in the biosynthesis of unique diterpenoids, and with this knowledge, we can further expand our understanding of diterpenoid metabolism in Cupressaceae and their potential role in plant defense.


Assuntos
Alquil e Aril Transferases/metabolismo , Chamaecyparis/enzimologia , Diterpenos/metabolismo , Proteínas de Plantas/metabolismo , Chamaecyparis/metabolismo , Clonagem Molecular , Escherichia coli , Perfilação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Redes e Vias Metabólicas , Organismos Geneticamente Modificados
17.
Plant Signal Behav ; 16(4): 1885906, 2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33570442

RESUMO

Terpene trilactones (TTLs) are the main medicinal compounds of Ginkgo biloba. Levopimaradiene synthase (LPS) is the crucial enzyme that catalyzes TTLs biosynthesis in G. biloba. In this study, a novel LPS gene (designated as GbLPS2) was cloned from G. biloba leaves. The open reading frame of GbLPS2 gene was 2520 bp in length, encoding a predicted polypeptide of 840 amino acids. Phylogenetic analysis revealed that the GbLPS2 was highly homologous with reported LPS proteins in other plants. On the basis of the genomic DNA (gDNA) template, a 4308 bp gDNA sequence of GbLPS2 and a 913 bp promoter sequence were amplified. Cis-acting elements in promoter analysis indicated that GbLPS2 could be regulated by methyl jasmonate (MeJA) and abscisic acid (ABA). Tissue-specific expression analysis revealed that GbLPS2 was mainly expressed in roots and ovulate strobilus. MeJA treatment could significantly induce the expression level of GbLPS2 and increase the content of TTLs. This study illustrates the structure and the tissue-specific expression pattern of GbLPS2 and demonstrates that exogenous hormones regulated the expression of GbLPS2 and TTL content in G. biloba. Our results provide a target gene for the enhancement of TTL content in G. biloba via genetic engineering.


Assuntos
Alquil e Aril Transferases/genética , Vias Biossintéticas/genética , Genes de Plantas , Ginkgo biloba/enzimologia , Ginkgo biloba/genética , Lactonas/metabolismo , Terpenos/metabolismo , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ginkgo biloba/efeitos dos fármacos , Especificidade de Órgãos/genética , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
J Biol Chem ; 296: 100444, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33617883

RESUMO

Unrepaired DNA-protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein cross-links can be cleaved into DNA-peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/farmacologia , Cromatografia Líquida/métodos , DNA/química , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Desoxiadenosinas/química , Desoxiadenosinas/metabolismo , Desoxiguanosina/metabolismo , Dibrometo de Etileno/química , Humanos , Cinética , Estrutura Molecular , Mutação , Nucleotídeos/genética , Peptídeos/genética , Espectrometria de Massas em Tandem/métodos
19.
J Clin Invest ; 131(5)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33444290

RESUMO

Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.


Assuntos
Ataxia/metabolismo , Indenos/farmacologia , Nefropatias/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Doenças Mitocondriais/metabolismo , Debilidade Muscular/metabolismo , Podócitos/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirazóis/farmacologia , Ubiquinona/deficiência , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Ataxia/tratamento farmacológico , Ataxia/genética , Ataxia/patologia , Sistemas de Liberação de Medicamentos , Células HEK293 , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/genética , Nefropatias/patologia , Peroxidação de Lipídeos/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Debilidade Muscular/tratamento farmacológico , Debilidade Muscular/genética , Debilidade Muscular/patologia , Podócitos/patologia , Proteínas Proto-Oncogênicas B-raf/genética , RNA-Seq , Ubiquinona/genética , Ubiquinona/metabolismo
20.
Biochem Biophys Res Commun ; 536: 107-114, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33387748

RESUMO

Sesquiterpene synthases catalyse cyclisation of farnesyl pyrophosphate to produce diverse sesquiterpenes. Despite utilising the same substrate and exhibiting significant sequence and structural homology, these enzymes form different products. Previous efforts were based on identifying the effect of divergent residues present at the catalytic binding pocket on the product specificity of these enzymes. However, the rationales deduced for the product specificity from these studies were not generic enough to be applicable to other phylogenetically distant members of this family. To address this problem, we have developed a novel approach combining sequence, structural and dynamical information of plant sesquiterpene synthases (SSQs) to predict product modulating residues (PMRs). We tested this approach on the SSQs with known PMRs and also on sesquisabinene synthase 1 (SaSQS1), a SSQ from Indian sandalwood. Our results show that the dynamical sectors of SSQs obtained from molecular dynamics simulation and their hydrophobicity and vicinity indices together provide leads for the identification of PMRs. The efficacy of the technique was tested on SaSQS1 using mutagenesis. To the best of our knowledge, this is a first technique of this kind which provides cues on PMRs of SSQs, with divergent phylogenetic relationship.


Assuntos
Alquil e Aril Transferases/metabolismo , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/química , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Santalum/enzimologia , Sesquiterpenos/química
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