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1.
ACS Chem Biol ; 17(5): 1226-1238, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35446557

RESUMO

Natural drimane-type sesquiterpenes, including drimenol, display diverse biological activities. These active compounds are distributed in plants and fungi; however, their accumulation in bacteria remains unknown. Consequently, bacterial drimane-type sesquiterpene synthases remain to be characterized. Here, we report five drimenol synthases (DMSs) of marine bacterial origin, all belonging to the haloacid dehalogenase (HAD)-like hydrolase superfamily with the conserved DDxxE motif typical of class I terpene synthases and the DxDTT motif found in class II diterpene synthases. They catalyze two continuous reactions: the cyclization of farnesyl pyrophosphate (FPP) into drimenyl pyrophosphate and dephosphorylation of drimenyl pyrophosphate into drimenol. Protein structure modeling of the characterized Aquimarina spongiae DMS (AsDMS) suggests that the FPP substrate is located within the interdomain created by the DDxxE motif of N-domain and DxDTT motif of C-domain. Biochemical analysis revealed two aspartate residues of the DDxxE motif that might contribute to the capture of the pyrophosphate moiety of FPP inside the catalytic site of AsDMS, which is essential for efficient cyclization and subsequent dephosphorylation reactions. The middle aspartate residue of the DxDTT motif is also critical for cyclization. Thus, AsDMS utilizes both motifs in the reactions. Remarkably, the unique protein architecture of AsDMS, which is characterized by the fusion of a HAD-like domain (N-domain) and a terpene synthase ß domain (C-domain), significantly differentiates this new enzyme. Our findings of the first examples of bacterial DMSs suggest the biosynthesis of drimane sesquiterpenes in bacteria and shed light on the divergence of the structures and functions of terpene synthases.


Assuntos
Alquil e Aril Transferases , Sesquiterpenos , Ácido Aspártico , Difosfatos , Genes Bacterianos , Sesquiterpenos Policíclicos , Sesquiterpenos/química
2.
Commun Biol ; 4(1): 1098, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535749

RESUMO

Intensive gene annotation has revealed many functional and regulatory elements in the human genome. Although eukaryotic protein-coding genes are generally transcribed into monocistronic mRNAs, recent studies have discovered additional short open reading frames (sORFs) in mRNAs. Here, we performed proteogenomic data mining for hidden proteins categorized into sORF-encoded polypeptides (SEPs) in human cancers. We identified a new SEP-encoding overlapping sORF (oORF) on the cell polarity determinant Scribble (SCRIB) that is considered a proto-oncogene with tumor suppressor function in Hippo-YAP/TAZ, MAPK/ERK, and PI3K/Akt/mTOR signaling. Reanalysis of clinical human proteomic data revealed translational dysregulation of both SCRIB and its oORF, oSCRIB, during carcinogenesis. Biochemical analyses suggested that the translatable oSCRIB constitutively limits the capacity of eukaryotic ribosomes to translate the downstream SCRIB. These findings provide a new example of cis-regulatory oORFs that function as a ribosomal roadblock and potentially serve as a fail-safe mechanism to normal cells for non-excessive downstream gene expression, which is hijacked in cancer.


Assuntos
Proteínas de Membrana/genética , Fases de Leitura Aberta/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo
3.
Nat Commun ; 11(1): 5664, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199711

RESUMO

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.


Assuntos
Biocatálise , Glucosiltransferases/metabolismo , Ácido Glucurônico/metabolismo , Saponinas/biossíntese , Vias Biossintéticas , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosilação , Glycyrrhiza uralensis/genética , Ácido Glicirrízico/metabolismo , Funções Verossimilhança , Lotus/genética , Filogenia , Saccharomyces cerevisiae/metabolismo , Saponinas/química , Glycine max/genética , Especificidade por Substrato , Triterpenos/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo
4.
J Nat Prod ; 82(12): 3311-3320, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31774676

RESUMO

Pentacyclic triterpenes may be active agents and provide a rich natural resource of promising compounds for drug development. The inhibitory activities of 29 natural oleanane and ursane pentacyclic triterpenes were evaluated against four major enzymes involved in the inflammatory process: 5-LOX, 15-LOX-2, COX-1, and COX-2. It was found that 3-O-acetyl-ß-boswellic acid potently inhibited human 15-LOX-2 (IC50 = 12.2 ± 0.47 µM). Analysis of the structure-activity relationships revealed that the presence of a hydroxy group at position 24 was beneficial in terms of both 5-LOX and COX-1 inhibition. Notably, the introduction of a carboxylic acid group at position 30 was important for dual 5-LOX/COX inhibitory activity; furthermore, its combination with a carbonyl group at C-11 considerably increased 5-LOX inhibition. Also, the presence of an α-hydroxy group at C-2 or a carboxylic acid group at C-23 markedly suppressed the 5-LOX activity. The present findings reveal that the types and configurations of polar moieties at positions C-2, -3, -11, -24, and -30 are important structural aspects of pentacyclic triterpenes for their potential as anti-inflammatory lead compounds.


Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Lipoxigenase/farmacologia , Triterpenos Pentacíclicos/farmacologia , Inibidores de Ciclo-Oxigenase/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Inibidores de Lipoxigenase/química , Triterpenos Pentacíclicos/química , Relação Estrutura-Atividade
5.
Plant J ; 99(6): 1127-1143, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31095780

RESUMO

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins.


Assuntos
Glicosiltransferases/metabolismo , Glycyrrhiza uralensis/enzimologia , Ácido Glicirrízico/metabolismo , Arginina/química , Arginina/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Glicosiltransferases/química , Glicosiltransferases/genética , Glycyrrhiza uralensis/genética , Glycyrrhiza uralensis/metabolismo , Ácido Glicirrízico/química , Cinética , Simulação de Acoplamento Molecular , Mutação , Filogenia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Medicinais/enzimologia , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Saponinas/análise , Transcriptoma , Triterpenos/química , Triterpenos/metabolismo , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/metabolismo
6.
Plant Cell Physiol ; 59(4): 792-805, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29401289

RESUMO

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.


Assuntos
Genes de Plantas , Variação Genética , Glycine max/genética , Proteínas de Plantas/genética , Saponinas/genética , Açúcares/metabolismo , Alelos , Sequência de Aminoácidos , Estudos de Associação Genética , Teste de Complementação Genética , Glicosiltransferases/metabolismo , Mutação/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Saponinas/química , Saponinas/metabolismo , Transgenes
7.
Plant J ; 94(2): 327-339, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29441718

RESUMO

Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria. Using an Escherichia coli mutant lacking ppGpp, CmRSH4b was demonstrated to have ppGpp synthetase activity. Expression analysis of a green fluorescent protein-fused protein indicated that CmRSH4b localizes to the chloroplast, and overexpression of the CmRSH4b gene resulted in a decrease of chloroplast rRNA synthesis concomitant with growth inhibition and reduction of chloroplast size. Biochemical analyses using C. merolae cell lysates or purified recombinant proteins revealed that ppGpp inhibits bacteria-type RNA polymerase-dependent chloroplast rRNA synthesis as well as a chloroplast guanylate kinase. These results suggest that CmRSH4b-dependent ppGpp synthesis in chloroplasts is an important regulator of chloroplast rRNA transcription. Nuclear and mitochondrial rRNA transcription were both reduced by TOR inhibition, suggesting that the biogeneses of the three independent ribosome systems are interconnected by TOR in plant cells.


Assuntos
Proteínas de Algas/metabolismo , Cloroplastos/metabolismo , Ligases/genética , RNA de Cloroplastos/metabolismo , RNA Ribossômico/metabolismo , Rodófitas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Algas/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ligases/metabolismo
8.
Biosci Biotechnol Biochem ; 78(6): 1022-5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25036129

RESUMO

The ppGpp-signaling system functions in plant chloroplasts. In bacteria, a negative effect of ppGpp on adenylosuccinate synthetase (AdSS) has been suggested. Our biochemical analysis also revealed rice AdSS homologs are apparently sensitive to ppGpp. However, further investigation clarified that this phenomenon is cancelled by the high substrate affinity to the enzymes, leading to a limited effect of ppGpp on adenylosuccinate synthesis.


Assuntos
Adenilossuccinato Sintase/metabolismo , Guanosina Tetrafosfato/farmacologia , Oryza/enzimologia , Purinas/biossíntese , Bacillus subtilis/enzimologia , Escherichia coli/enzimologia , Guanosina Tetrafosfato/química , Cinética , Oryza/metabolismo , Homologia de Sequência de Aminoácidos
9.
J Biol Chem ; 289(22): 15631-41, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24722991

RESUMO

The guanosine 3',5'-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 µM relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 µM. The IC50 of ppGpp for GKpm was ∼10 µM. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.


Assuntos
Bactérias/enzimologia , Cloroplastos/enzimologia , Guanosina Tetrafosfato/metabolismo , Guanilato Quinases/metabolismo , Ligases/metabolismo , Plantas/enzimologia , Arabidopsis/enzimologia , Arabidopsis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Bactérias/genética , Sequência de Bases , Cloroplastos/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Guanilato Quinases/genética , Ligases/genética , Dados de Sequência Molecular , Oryza/enzimologia , Oryza/genética , Pisum sativum/enzimologia , Pisum sativum/genética , Plantas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologia , Synechococcus/enzimologia , Synechococcus/genética
10.
Plant Mol Biol ; 78(1-2): 185-96, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22108865

RESUMO

Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.


Assuntos
Cloroplastos/genética , Guanosina Tetrafosfato/metabolismo , Elongação Traducional da Cadeia Peptídica/genética , Proteínas de Plantas/genética , Antibacterianos/farmacologia , Radioisótopos de Carbono , Cloroplastos/efeitos dos fármacos , Cloroplastos/metabolismo , Relação Dose-Resposta a Droga , Ácido Fusídico/farmacologia , Guanosina Difosfato/metabolismo , Guanosina Difosfato/farmacologia , Guanosina Tetrafosfato/farmacologia , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Leucina/genética , Leucina/metabolismo , Pisum sativum/genética , Pisum sativum/metabolismo , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Poli U/genética , RNA Mensageiro/genética , Tioestreptona/farmacologia
11.
Protein Expr Purif ; 73(1): 15-22, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20304073

RESUMO

Wheat-embryo cell-free protein expression system allows efficient production of a wide variety of proteins. Homogeneity of the end products is an important characteristic of an advanced cell-free system that will be used in a field of protein science such as structural biology. A translation enhancer such as the omega sequence derived from tobacco mosaic virus, that allows cap-independent translation of the mRNA in the cell-free system, is required for low-cost preparation of template mRNAs in the cell-free translation system. However, the use of translational enhancers often leads to unexpected byproducts. Several AUU codons in the omega sequence can potentially function as translation initiators. We confirmed that the in-frame AUU in the omega sequence functions as a non-canonical start codon and results in the extension of the N-terminus of the target protein in some cases. Investigation of the selectivity of non-canonical initiation codon under the control of omega sequence in the wheat-embryo cell-free system revealed that seven non-AUG codons, CUG, AUA, AUU, GUG, ACG, AUC, and UUG, are recognized as translation initiators. We found that the introduction of an in-frame stop codon just upstream of the target open reading frame is an efficient way to avoid unexpected byproducts. This minor but effective modification facilitates production of homogeneous proteins within the wheat-embryo cell-free protein expression system at the preparative scale.


Assuntos
Códon/genética , Proteínas de Plantas/genética , Biossíntese de Proteínas/genética , Sementes/química , Sítio de Iniciação de Transcrição , Triticum/química , Sequência de Aminoácidos , Sequência de Bases , Sistema Livre de Células , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Oryza , Extratos Vegetais/metabolismo , Proteínas de Plantas/biossíntese , Pirofosfatases/biossíntese , Pirofosfatases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
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