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1.
Chem Commun (Camb) ; 57(51): 6308-6311, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34075974

RESUMO

Here, we describe the identification of PARP1/2 as direct binding proteins of andrographolide (Andro) using protein microarray, surface plasmon resonance (SPR), and enzyme activity assays. We then evaluated the proliferation inhibition, apoptosis, and cell migration effects of Andro on the MDA-MB-436 cell line in vitro. The final biological evaluation confirmed that Andro was a highly effective single agent in the MDA-MB-436 xenograft model and had a low hERG-mediated cardiac toxicity. Therefore, Andro represents the first natural product, non-amide member of a novel nanomolar-potency PARP1/2 inhibitor family.


Assuntos
Diterpenos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diterpenos/farmacologia , Diterpenos/uso terapêutico , Ensaios Enzimáticos , Humanos , Cinética , Camundongos , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ftalazinas/metabolismo , Ftalazinas/farmacologia , Piperazinas/metabolismo , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/análise , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/análise , Poli(ADP-Ribose) Polimerases/química , Análise Serial de Proteínas , Razão Sinal-Ruído , Ressonância de Plasmônio de Superfície , Transplante Heterólogo
2.
Theranostics ; 11(15): 7199-7221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34158845

RESUMO

Triptolide, an abietane-type diterpenoid isolated from Tripterygium wilfordii Hook. F., has significant pharmacological activity. Research results show that triptolide has obvious inhibitory effects on many solid tumors. Therefore, triptolide has become one of the lead compounds candidates for being the next "blockbuster" drug, and multiple triptolide derivatives have entered clinical research. An increasing number of researchers have developed triptolide synthesis methods to meet the clinical need. To provide new ideas for researchers in different disciplines and connect different disciplines with researchers aiming to solve scientific problems more efficiently, this article reviews the research progress made with analyzes of triptolide pharmacological activity, biosynthetic pathways, and chemical synthesis pathways and reported in toxicological and clinical studies of derivatives over the past 20 years, which have laid the foundation for subsequent researchers to study triptolide in many ways.


Assuntos
Antineoplásicos Fitogênicos , Diterpenos , Fenantrenos , Tripterygium , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/uso terapêutico , Diterpenos/síntese química , Diterpenos/metabolismo , Diterpenos/uso terapêutico , Compostos de Epóxi/síntese química , Compostos de Epóxi/metabolismo , Compostos de Epóxi/uso terapêutico , Humanos , Fenantrenos/síntese química , Fenantrenos/metabolismo , Fenantrenos/uso terapêutico , Tripterygium/química , Tripterygium/metabolismo
3.
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
5.
Artigo em Inglês | MEDLINE | ID: mdl-34052752

RESUMO

Detailed metabolic profiling of needles of five Pinus species was investigated using complementary HPLC-MS/MS techniques together with supervised and unsupervised chemometric tools. This resulted in putative identification of 44 compounds belonging to flavonoids, phenolics, lignans, diterpenes and fatty acids. Unsupervised principal component analysis showed that differences were maintained across the metabolites characteristic of each Pinus species, are mainly related to di-O-p-coumaroyltrifolin, p-coumaroyl quinic acid derivative, arachidonic acid, hydroxypalmitic acid, isopimaric acid and its derivative. A supervised Partial Least Squares regression analysis was performed to correlate HPLC-MS/MS profiles with the variation observed in the in vitro anticholinesterase, antiaging and anti-diabetic potential. All investigated Pinus extracts exerted their antiaging activity via increasing telomerase and TERT levels in normal human melanocytes cells compared to the control (untreated cells). Profound inhibition activities of acetylcholinesterase and dipeptidyl peptidase-4 were also observed with P. pinea and P. canariensis extracts having comparable antidiabetic activities to sitagliptin as a standard antidiabetic drug. Our findings suggested that pine needles are a good source of phenolics and diterpenoids that have possible health promoting activities in management and alleviation of diabetic conditions and Alzheimer disease.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metaboloma/fisiologia , Pinus , Espectrometria de Massas em Tandem/métodos , Inibidores da Colinesterase/análise , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Diterpenos/análise , Diterpenos/química , Diterpenos/metabolismo , Flavonoides/análise , Flavonoides/química , Flavonoides/metabolismo , Hipoglicemiantes/análise , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Metabolômica , Pinus/química , Pinus/metabolismo , Extratos Vegetais/química , Substâncias Protetoras/análise , Substâncias Protetoras/química , Substâncias Protetoras/metabolismo , Extração em Fase Sólida/métodos
6.
Plant Cell ; 33(2): 290-305, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793769

RESUMO

Plants can contain biosynthetic gene clusters (BGCs) that nominally resemble those found in microbes. However, while horizontal gene transmission is often observed in microbes, plants are limited to vertical gene transmission, implying that their BGCs may exhibit distinct inheritance patterns. Rice (Oryza sativa) contains two unlinked BGCs involved in diterpenoid phytoalexin metabolism, with one clearly required for momilactone biosynthesis, while the other is associated with production of phytocassanes. Here, in the process of elucidating momilactone biosynthesis, genetic evidence was found demonstrating a role for a cytochrome P450 (CYP) from the other "phytocassane" BGC. This CYP76M8 acts after the CYP99A2/3 from the "momilactone" BGC, producing a hemiacetal intermediate that is oxidized to the eponymous lactone by a short-chain alcohol dehydrogenase also from this BGC. Thus, the "momilactone" BGC is not only incomplete, but also fractured by the need for CYP76M8 to act in between steps catalyzed by enzymes from this BGC. Moreover, as supported by similar activity observed with orthologs from the momilactone-producing wild-rice species Oryza punctata, the presence of CYP76M8 in the other "phytocassane" BGC indicates interdependent evolution of these two BGCs, highlighting the distinct nature of BGC assembly in plants.


Assuntos
Evolução Biológica , Vias Biossintéticas/genética , Diterpenos/metabolismo , Família Multigênica , Oryza/genética , Diterpenos/química , Regulação da Expressão Gênica de Plantas , Oxirredução , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
7.
J Ind Microbiol Biotechnol ; 48(1-2)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33928347

RESUMO

Forskolin, a class of labdane-type diterpenoid, has significant medicinal value in anticancer, antiasthmatic, antihypertensive, and heart-strengthening treatments. The main source of natural forskolin is its extraction from the cork tissue of the root of Coleus forskohlii. However, conventional modes of extraction pose several challenges. In recent years, the construction of microbial cell factories to produce medicinal natural products via synthetic biological methods has effectively solved the current problems and is a research hotspot in this field. This review summarizes the recent progress in the heterologous synthesis of forskolin via synthetic biological technology, analyzes the current challenges, and proposes corresponding strategies.


Assuntos
Colforsina/metabolismo , Colforsina/química , Diterpenos/química , Diterpenos/metabolismo
8.
BMC Plant Biol ; 21(1): 191, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879061

RESUMO

BACKGROUND: Cytochrome P450s (P450s) are enzymes that play critical roles in the biosynthesis of physiologically important compounds across all organisms. Although they have been characterised in a large number of plant species, no information relating to these enzymes are available from the genus Fontainea (family Euphorbiaceae). Fontainea is significant as the genus includes species that produce medicinally significant epoxy-tigliane natural products, one of which has been approved as an anti-cancer therapeutic. RESULTS: A comparative species leaf metabolome analysis showed that Fontainea species possess a chemical profile different from various other plant species. The diversity and expression profiles of Fontainea P450s were investigated from leaf and root tissue. A total of 103 and 123 full-length P450 genes in Fontainea picrosperma and Fontainea venosa, respectively (and a further 127/125 partial-length) that were phylogenetically classified into clans, families and subfamilies. The majority of P450 identified are most active within root tissue (66.2% F. picrosperma, 65.0% F. venosa). Representatives within the CYP71D and CYP726A were identified in Fontainea that are excellent candidates for diterpenoid synthesis, of which CYP726A1, CYP726A2 and CYP71D1 appear to be exclusive to Fontainea species and were significantly more highly expressed in root tissue compared to leaf tissue. CONCLUSION: This study presents a comprehensive overview of the P450 gene family in Fontainea that may provide important insights into the biosynthesis of the medicinally significant epoxy-tigliane diterpenes found within the genus.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Diterpenos/metabolismo , Euphorbiaceae/genética , Genes de Plantas , Proteínas de Plantas/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Euphorbiaceae/enzimologia , Euphorbiaceae/metabolismo , Família Multigênica , Proteínas de Plantas/metabolismo
9.
Angew Chem Int Ed Engl ; 60(25): 14163-14170, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-33780586

RESUMO

A new bicyclic diterpenoid, benditerpenoic acid, was isolated from soil-dwelling Streptomyces sp. (CL12-4). We sequenced the bacterial genome, identified the responsible biosynthetic gene cluster, verified the function of the terpene synthase, and heterologously produced the core diterpene. Comparative bioinformatics indicated this Streptomyces strain is phylogenetically unique and possesses nine terpene synthases. The absolute configurations of the new trans-fused bicyclo[8.4.0]tetradecanes were achieved by extensive spectroscopic analyses, including Mosher's analysis, J-based coupling analysis, and computations based on sparse NMR-derived experimental restraints. Interestingly, benditerpenoic acid exists in two distinct ring-flipped bicyclic conformations with a rotational barrier of ≈16 kcal mol-1 in solution. The diterpenes exhibit moderate antibacterial activity against Gram-positive bacteria including methicillin and multi-drug resistant Staphylococcus aureus. This is a rare example of an eunicellane-type diterpenoid from bacteria and the first identification of a diterpene synthase and biosynthetic gene cluster responsible for the construction of the eunicellane scaffold.


Assuntos
Antibacterianos/biossíntese , Diterpenos/metabolismo , Descoberta de Drogas , Streptomyces/química , Antibacterianos/química , Antibacterianos/farmacologia , Diterpenos/química , Diterpenos/farmacologia , Conformação Molecular , Streptomyces/genética , Streptomyces/metabolismo
10.
Plant Mol Biol ; 106(1-2): 145-156, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33694047

RESUMO

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.


Assuntos
Diterpenos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Linhagem Celular , Compostos de Epóxi/metabolismo , Proteínas de Membrana Transportadoras/química , Mutagênese/genética , Filogenia , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Estabilidade Proteica , Protoplastos/metabolismo , Tabaco/genética , Transcrição Genética , Tripterygium/genética
11.
Plant Cell ; 33(5): 1748-1770, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33561278

RESUMO

The native diploid tobacco Nicotiana attenuata produces abundant, potent anti-herbivore defense metabolites known as 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 family-1 UGT members. The transcript levels of three UGTs were highly correlated with the transcript levels two key HGL-DTG biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLS's role in HGL-DTG biosynthesis was confirmed by virus-induced gene silencing. Silencing the Uridine diphosphate (UDP)-rhamnosyltransferase gene UGT91T1 demonstrated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. Plants with stable silencing of UGT74P3 and UGT74P5 were severely developmentally deformed, pointing to a phytotoxic effect of the aglycone. The application of synthetic 17-HGL and silencing of the UGTs in HGL-DTG-free plants confirmed this phytotoxic effect. Feeding assays with tobacco hornworm (Manduca sexta) larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation of 17-HGL is therefore a critical step that contributes to the resulting metabolites' defensive function and solves the autotoxicity problem of this potent chemical defense.


Assuntos
Monoterpenos Acíclicos/metabolismo , Diterpenos/metabolismo , Glicosídeos/metabolismo , Tabaco/metabolismo , Monoterpenos Acíclicos/química , Animais , Vias Biossintéticas , Inativação Gênica , Glicosilação , Glicosiltransferases/metabolismo , Herbivoria , Larva/fisiologia , Manduca/fisiologia , Metabolômica , Necrose , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/metabolismo
12.
Microb Cell Fact ; 20(1): 29, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33530990

RESUMO

BACKGROUND: Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis. RESULTS: We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20. CONCLUSIONS: We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.


Assuntos
Vias Biossintéticas , Diterpenos/metabolismo , Ácido Mevalônico/metabolismo , Saccharomyces cerevisiae/metabolismo , Diterpenos/química , Saccharomyces cerevisiae/crescimento & desenvolvimento
13.
Acc Chem Res ; 54(5): 1131-1142, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33544578

RESUMO

Halogenated natural products number in the thousands, but only in rare cases are the evolutionary advantages conferred by the halogens understood. We set out to investigate the lissoclimide family of cytotoxins, which includes several chlorinated members, because of our long-standing interest in the synthesis of chlorinated secondary metabolites.Our initial success in this endeavor was a semisynthesis of chlorolissoclimide (CL) from the commercially available sesquiterpenoid sclareolide. Featuring a highly selective and efficient-and plausibly biomimetic-C-H chlorination, we were able to access enough CL for collaborative studies, including X-ray cocrystallography with the eukaryotic ribosome. Through this experiment, we learned that CL's chlorine atom engages in a novel halogen-π dispersion interaction with a neighboring nucleobase in the ribosome E-site.Owing to the limitations of our semisynthesis approach, we established an analogue-oriented approach to access numerous lissoclimide compounds to both improve our understanding of structure-activity relationships and to learn more about the halogen-π interaction. In the course of these studies, we made over a dozen lissoclimide-like compounds, the most interesting of which contained chlorine-bearing carbons with unnatural configurations. Rationalizing the retained potency of these compounds that appeared to be a poor fit for the lissoclimide binding pocket, we came to realize that the chlorine atoms would engage in these same halogen-π interactions even at the expense of a chair to twist-boat conformational change, which also permitted the compounds to fit in the binding site.Finally, because neither of the first two approaches could easily access the most potent natural lissoclimides, we designed a synthesis that took advantage of rarely used terminal epoxides to initiate polyene cyclizations. In this case, the chlorine atom was incorporated early and helped control the stereochemical outcome of the key step.Over the course of this project, three different synthesis approaches were designed and executed, and our ability to access numerous lissoclimides fueled a range of collaborative biological studies. Further, chlorine played impactful roles throughout various aspects of both synthesis and biology. We remain inspired to learn more about the mechanism of action of these compounds and to deeply investigate the potentially valuable halogen-π dispersion interaction in the context of small molecule/nucleic acid binding. In that context, our work offers an instance wherein we might have gained a rudimentary understanding of the evolutionary importance of the halogen in a halogenated natural product.


Assuntos
Produtos Biológicos/metabolismo , Cloro/metabolismo , Diterpenos/metabolismo , Succinimidas/metabolismo , Produtos Biológicos/síntese química , Produtos Biológicos/química , Cloro/química , Cristalografia por Raios X , Diterpenos/síntese química , Diterpenos/química , Halogenação , Modelos Moleculares , Conformação Molecular , Succinimidas/síntese química , Succinimidas/química
14.
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
15.
Science ; 371(6526): 255-260, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33446550

RESUMO

Many plant specialized metabolites function in herbivore defense, and abrogating particular steps in their biosynthetic pathways frequently causes autotoxicity. However, the molecular mechanisms underlying their defense and autotoxicity remain unclear. Here, we show that silencing two cytochrome P450s involved in diterpene biosynthesis in the wild tobacco Nicotiana attenuata causes severe autotoxicity symptoms that result from the inhibition of sphingolipid biosynthesis by noncontrolled hydroxylated diterpene derivatives. Moreover, the diterpenes' defensive function is achieved by inhibiting herbivore sphingolipid biosynthesis through postingestive backbone hydroxylation products. Thus, by regulating metabolic modifications, tobacco plants avoid autotoxicity and gain herbivore defense. The postdigestive duet that occurs between plants and their insect herbivores can reflect the plant's solutions to the "toxic waste dump" problem of using potent chemical defenses.


Assuntos
Diterpenos/metabolismo , Glucosídeos/biossíntese , Herbivoria , Manduca/fisiologia , Esfingolipídeos/biossíntese , Tabaco/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Manduca/enzimologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Tabaco/enzimologia
16.
J Am Chem Soc ; 143(10): 3794-3807, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33496585

RESUMO

Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme's specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.


Assuntos
Alquil e Aril Transferases/metabolismo , Engenharia de Proteínas , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Biocatálise , Cristalografia por Raios X , Ciclização , Diterpenos/química , Diterpenos/metabolismo , Mutagênese Sítio-Dirigida , Conformação Proteica , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Especificidade por Substrato
17.
Sci Rep ; 11(1): 333, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431904

RESUMO

Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize (Zea mays) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 (anther ear 2), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.


Assuntos
Antifúngicos/metabolismo , Diterpenos/metabolismo , Microbiota , Raízes de Plantas/microbiologia , Zea mays/metabolismo , Zea mays/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Rizosfera
18.
Mar Drugs ; 19(1)2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33467112

RESUMO

Chemical investigation of a Red Sea Spongia sp. led to the isolation of four new compounds, i.e., 17-dehydroxysponalactone (1), a carboxylic acid, spongiafuranic acid A (2), one hydroxamic acid, spongiafuranohydroxamic acid A (3), and a furanyl trinorsesterpenoid 16-epi-irciformonin G (4), along with three known metabolites (-)-sponalisolide B (5), 18-nor- 3,17-dihydroxy-spongia-3,13(16),14-trien-2-one (6), and cholesta-7-ene-3ß,5α-diol-6-one (7). The biosynthetic pathway for the molecular skeleton of 1 and related compounds was postulated for the first time. Anti-inflammatory activity of these metabolites to inhibit superoxide anion generation and elastase release in N-formyl-methionyl-leucyl phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophil cells and cytotoxicity of these compounds toward three cancer cell lines and one human dermal fibroblast cell line were assayed. Compound 1 was found to significantly reduce the superoxide anion generation and elastase release at a concentration of 10 µM, and compound 5 was also found to display strong inhibitory activity against superoxide anion generation at the same concentration. Due to the noncytotoxic activity and the potent inhibitory effect toward the superoxide anion generation and elastase release, 1 and 5 can be considered to be promising anti-inflammatory agents.


Assuntos
Anti-Inflamatórios/metabolismo , Diterpenos/metabolismo , Poríferos/metabolismo , Terpenos/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Células Cultivadas , Diterpenos/química , Diterpenos/farmacologia , Humanos , Oceano Índico , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Poríferos/química , Terpenos/química , Terpenos/farmacologia
19.
BMC Complement Med Ther ; 21(1): 41, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478471

RESUMO

BACKGROUND: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. METHODS: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. RESULTS: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. CONCLUSION: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.


Assuntos
Alérgenos/química , Alérgenos/imunologia , COVID-19/tratamento farmacológico , COVID-19/imunologia , Odorantes , Perfumes/química , SARS-CoV-2/química , SARS-CoV-2/imunologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alérgenos/administração & dosagem , Alérgenos/uso terapêutico , Benzopiranos/química , Benzopiranos/metabolismo , Compostos de Benzil/química , Compostos de Benzil/metabolismo , Cinamatos/química , Cinamatos/metabolismo , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Perfumes/administração & dosagem , Perfumes/uso terapêutico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
20.
Nat Chem Biol ; 17(2): 205-212, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33106662

RESUMO

Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete momilactone biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in Nicotiana benthamiana, a heterologous plant host. This required us to solve a central problem: diminishing intermediate and product yields remain a bottleneck for multistep diterpene pathways. We increased intermediate and product titers by rerouting diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement in production versus rice). Pure momilactone B isolated from N. benthamiana inhibited germination and root growth in Arabidopsis thaliana, validating allelopathic activity. We demonstrated the broad utility of this approach by applying it to forskolin, a Hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement in production versus chloroplast expression).


Assuntos
Diterpenos/metabolismo , Lactonas/metabolismo , Plantas/metabolismo , Transdução de Sinais/genética , Alcenos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/genética , Colforsina/farmacologia , Citosol/metabolismo , Diterpenos/farmacologia , Ácido Mevalônico/metabolismo , Oryza/genética , Paclitaxel/biossíntese , Folhas de Planta/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Tabaco/metabolismo
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