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1.
Plant Physiol Biochem ; 164: 185-194, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34004556

RESUMO

Agarwood is the resinous portion of Aquilaria trees, and has been widely used as medicine and incense. Sesquiterpenes are the main chemical characteristic constituents of agarwood. Terpene synthase (TPS) is a critical enzyme responsible for biosynthesis of sesquiterpene compounds. However, limited information is available on genome-wide identification and characterization of the TPS family in Aquilaria trees. In this study, TPS gene family was identified and characterized in Aquilaria sinensis by bioinformatics methods. The expression of those genes was analyzed by RNA-seq and quantitative real-time PCR. Transcription factors regulating TPS gene expression were identified by yeast one-hybrid and dual-luciferase assay. In total, 26 AsTPS genes (AsTPS1-AsTPS26) were identified, which were classified into five subgroups. Many putative cis-elements putatively involved in stresses and phytohormones (especially jasmonic acid) were identified in the promoter regions of AsTPSs, suggesting that AsTPSs genes may be regulated by stresses and jasmonic acid. Expression analysis revealed seven TPS genes encoding sesquiterpene synthetases were induced by wounding and methyl jasmonic acid (MeJA), which may be related to sesquiterpene biosynthesis. By yeast one-hybrid screening, a ERF transcription factor AsERF1 was identified to interact with the AsTPS1 promoter. Subcellular localization analysis indicated AsERF1 was a nucleus-localized protein. Transient transfection of AsERF1 in leaves of Nicotiana benthamiana significantly enhanced the promoter activation of AsTPS1, suggesting AsERF1 may participate in sesquiterpene biosynthesis by regulating AsTPS1 expression. These data generated in this study provide a foundation for future studies on functional roles and regulation mechanisms of AsTPS in sesquiterpene biosynthesis and agarwood formation.


Assuntos
Alquil e Aril Transferases , Sesquiterpenos , Thymelaeaceae , Alquil e Aril Transferases/genética , Thymelaeaceae/genética , Fatores de Transcrição/genética
2.
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
3.
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
4.
Phytochemistry ; 184: 112672, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33524857

RESUMO

All land plants (embryophytes) must contain an ent-kaurene synthase (KS), as the ability to produce this olefin from ent-copalyl diphosphate (ent-CPP) is required for phytohormone biosynthesis. These KSs have frequently given rise to other class I diterpene synthases that catalyze distinct reactions for more specialized plant metabolism. Indeed, the prevalence of such gene duplication and neofunctionalization has obscured phylogenetic assignment of function. Here a pair of threonines is found to be conserved in all land plant KS involved in phytohormone biosynthesis, and their role in enzyme function investigated. Surprisingly, these threonines are not required, nor even particularly important for efficient production of ent-kaurene from ent-CPP. In addition, these threonines do not seem to affect protein structure or stability. Moreover, the absence of codon bias and positioning within an intron do not support a role in transcription or translation either. Despite their lack of apparent function, this pair of threonines are nevertheless completely conserved in all embryophyte KS from phytohormone biosynthesis. Thus, regardless of exact role, this serves as a diagnostic mark for such KS, enabling more confident distinction of these critical enzymes.


Assuntos
Alquil e Aril Transferases , Diterpenos do Tipo Caurano , Alquil e Aril Transferases/genética , Giberelinas , Filogenia , Reguladores de Crescimento de Plantas , Proteínas de Plantas/genética , Treonina
5.
Phytochemistry ; 185: 112687, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33588133

RESUMO

Chrysanthemum indicum has long been used in traditional Chinese medicine for its health-promoting benefits. Studies on C. indicum have mainly focused on the flowers. Terpenoid distribution in various parts of the plant and characterization of terpene synthases remain unclear. In this study, volatile metabolic profiling was performed to compare the composition and quantity of terpenoids distributed in the root, stem, leaf, flower bud and flower of C. indicum. The potential for extracting active ingredients from the root, stem, and leaf was also examined. In total, 17 monoterpenoids and 27 sesquiterpenoids were identified. Transcriptome data were used to clone two monoterpene synthases and two sesquiterpene synthases highly expressed in the root. The recombinant proteins of full-length and truncated versions of C. indicum terpene synthase (CiTPS1) produced α-pinene, but the truncated one was catalytically more efficient than the full-length version. No product could be detected when full-length version of CiTPS2 was used for catalyzing GPP, but the truncated one can produce a minor amount of α-pinene. CiTPS3 contributed to the production of three sesquiterpenoids, namely ß-farnesene, petasitene, and α-bisabolene. CiTPS4 acted as a difunctional enzyme, contributing to the production of four monoterpenoids and three sesquiterpenoids, including petasitene. The evidence suggests that petasitene and the genes responsible for its biosynthesis were first found in the genus Chrysanthemum. The present findings provide insights into the composition, formation, and regulation of these bioactive compounds.


Assuntos
Alquil e Aril Transferases , Chrysanthemum , Alquil e Aril Transferases/genética , Chrysanthemum/genética , Flores , Terpenos
6.
J Agric Food Chem ; 69(7): 2168-2178, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33566615

RESUMO

Amorpha-4,11-diene synthase (ADS) is the first committed enzyme in the biosynthesis of artemisinin. Artemisinin production by biobased fermentation is considered a reliable alternative pathway. Heterologously expressed ADS has been established to generate several minor products, including structural analogues of amorpha-4,11-diene, but their fate in fermentation is still unknown. Here, using chiral analysis, we found that ADS produces one of the analogues, amorpha-4-en-11-ol, as a pair of epimers. Labeling experiments revealed that ADS mutants yielded amorphene-type sesquiterpenes, indicating the co-occurrence of initial 1,6 and 1,10 cyclization of farnesyl diphosphate in a single enzyme. Interestingly, the immediate downstream oxidase CYP71AV1 had very low affinity to the side products of the recombinant ADS, including amorpha-4-en-7-ol, which is structurally similar to amorpha-4,11-diene. Our data uncover the complex catalytic mechanism of recombinant ADS and reveal a potential negative effect of the side products of recombinant ADS on the production of the artemisinin precursor in microbes.


Assuntos
Alquil e Aril Transferases , Artemisininas , Fabaceae , Sesquiterpenos , Alquil e Aril Transferases/genética , Oxirredutases
7.
Dermatol Ther ; 34(2): e14854, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33548109

RESUMO

Psoriasis is a chronic and recurrent inflammatory skin disorder driven by a complex cascade of inflammatory mediators. The present study focused on the potential clinical significance of PGGT1B in psoriasis development. The peripheral blood mononuclear cells (PBMCs) were isolated from 81 psoriasis patients and 84 healthy controls, and the expression levels of PGGT1B in PBMCs were examined by quantitative real-time polymerase chain reaction (RT-qPCR) methods. Furthermore, we tested the relationship between the level of PGGT1B in PBMCs and psoriasis severity. Also, we analyzed the potential significance of PGGT1B in psoriasis diagnosis. Finally, patients with psoriasis were divided into progressive and stable stage groups, and the differential expression of PGGT1B, TNF-α, IL-17, and IFN-γ between different phases were analyzed. PGGT1B was dramatically decreased in the psoriasis patients' PBMCs and negatively correlated with the Psoriasis Area and Severity Index (PASI). Moreover, receiver operating characteristics analysis showed the potential of differentially expressed PGGT1B in terms of distinguishing psoriasis patients from healthy controls. Finally, compared to the patients in the stable phase, PGGT1B was markedly reduced in patients' PBMCs in the progressive stage, while proinflammatory cytokines TNF-α and IL-17 were notably increased. PGGT1B was downregulated in psoriasis patients' PBMCs and may serve as a potential biomarker for the diagnosis and treatment of psoriasis.


Assuntos
Alquil e Aril Transferases/genética , Leucócitos Mononucleares , Psoríase , Estudos de Casos e Controles , Citocinas/genética , Humanos , Mediadores da Inflamação , Psoríase/diagnóstico , Psoríase/genética , Reação em Cadeia da Polimerase em Tempo Real
8.
Gene ; 773: 145417, 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33444679

RESUMO

Melissa officinalis (lemon balm) is a well-known pharmaceutical plant in traditional medicine around the world because of the high-value secondary metabolites. Nowadays, advances in computational biology and bioinformatics have opened new avenues to plant-based natural product drug discovery. Despite the pharmacological importance, there is low information about the genes encoding the important biosynthetic pathways related to the secondary metabolite in M. officinalis. In this study, the main genes related to the rosmarinic acid (RA) and terpenoid biosynthesis pathways were detected using transcriptome analysis. Furthermore, we isolated and characterized a novel M. officinalis Hydroxyphenylpyruvate reductase (HPPR) gene involved in RA biosynthesis pathway. An effective pipeline was used to generate 37,055 unigenes by evaluating 42,837,601 Illumina paired-end reads. Functional annotation of the unigenes revealed that 27,363 (73.84%) and 35,822 (96.67%) unigenes had significant similarity to identified proteins in the SwissProt and NR databases, respectively. Also, 10,062 (36.83%) out of 37,055 unigenes were assigned to 399 KEGG pathways. Since terpenes and RA are two prominent metabolites in this plant, the attention of this study has been on the pathways related to them. A total of 149 unigenes were found that are related to the terpenoids biosynthesis, including 75 unigenes involved in the methyl-erythritol phosphate and mevalonate pathway, terpenoid backbone biosynthesis genes, and 74 unigenes related to the terpene synthase. We also identified 144 and 30 unigenes that were associated with the biosynthesis of phenylpropanoid and the rosmarinic acid pathway. Consequently, this investigation can be a comprehensive and accurate transcriptome basis for further investigation in the metabolic engineering and detection of new genes and pathways in M. officinalis.


Assuntos
Cinamatos/metabolismo , Depsídeos/metabolismo , Melissa/genética , Terpenos/metabolismo , Transcriptoma/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Vias Biossintéticas/genética , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Melissa/metabolismo , Anotação de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo
9.
J Agric Food Chem ; 69(4): 1413-1429, 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33481572

RESUMO

Terpenes and their derivatives are important biomarkers of grape quality as they contribute to the flavor and aroma of grapes. However, the molecular basis of terpene biosynthesis throughout the grapevine phenological developmental cycle remains elusive. Our current study investigates the free and bound terpene biosynthesis of berries at different phenological stages from preveraison to harvest. Detailed gene expression (transcriptomics) analysis, terpenoid volatile production by gas chromatography-mass spectrometry (GC-MS), and in planta transient expression were employed. Our results show that concentrations of most individual terpenes at different stages are distinctive and increase from preveraison to the veraison stage followed by a decrease from veraison to maturity. The combined transcriptomic analysis and terpene profiling revealed that 22 genes belonging to the MEP pathway and multiple classes of transcription factor family members including bHLH and several hormone biosynthesis- or signaling-related genes likely participate in the regulation of terpenoid biosynthesis according to their specific expression patterns in berries. Quantitative real-time polymerase chain expression analysis of 8 key differentially expressed genes in MEP pathways and further 12 randomly selected genes was performed during 8 sampling stages and validated the RNA-seq-derived expression profiles. To further confirm the function of a subset of the differentially expressed genes, we investigated the effects of combined overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (VvDXS1-LOC100249323), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (VvDXR-LOC100248516), and terpene synthase (VvTPS56-LOC100266449) on the production of terpenes by transient overexpression in Nicotiana benthamiana leaves. The overall developmental patterns of total terpenes and gene expression profiles will help guide the functional analyses of further candidate genes important for terpene biosynthesis of grape as well as identifying the master transcriptional and hormonal regulators of this pathway in the future.


Assuntos
Alquil e Aril Transferases/metabolismo , Eritritol/análogos & derivados , Aromatizantes/metabolismo , Frutas/química , Frutas/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Fosfatos Açúcares/metabolismo , Terpenos/metabolismo , Vitis/genética , Alquil e Aril Transferases/genética , Eritritol/metabolismo , Aromatizantes/química , Frutas/genética , Frutas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Terpenos/química , Vitis/química , Vitis/crescimento & desenvolvimento , Vitis/metabolismo
10.
J Plant Physiol ; 258-259: 153358, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33453433

RESUMO

Silk tree, Albizia julibrissin Duraz, is an old ornamental plant and extensively cultivated in Asia. Previous works have discovered that the terpenoids were the dominating compounds in the floral VOC of A. julibrissin, however the biosynthesis of these terpenoids was poorly understood so far. Here, 11 terpene synthase genes (TPSs) were identified by transcriptome sequencing that fell into TPS-a, TPS-b and TPS-g subfamilies. The enzymatic activity tests showed that five genes were functional: AjTPS2 was a sesquiterpene synthase and produced α-farnesene and (Z, E)-ß-farnesene; AjTPS5 was able to catalyze the formation of five monoterpenes and nine sesquiterpenes; AjTPS7, AjTPS9 and AjTPS10 were dedicated monoterpene synthases, as AjTPS7 and AjTPS10 formed the single product ß-ocimene and linalool, respectively, and AjTPS9 produced γ-terpinene with other three monoterpenes. More importantly, the main catalytic products of the characterized AjTPSs were consistent with the terpenoids observed in A. julibrissin volatiles. Combining terpene chemistry, TPSs biochemical activities and gene expression analysis, we demonstrate that AjTPS2, AjTPS5, AjTPS7, AjTPS9 and AjTPS10 are responsible for the volatile terpenoids biosynthesis in A. julibrissin.


Assuntos
Albizzia/genética , Alquil e Aril Transferases/genética , Proteínas de Plantas/genética , Terpenos/metabolismo , Albizzia/enzimologia , Albizzia/metabolismo , Alquil e Aril Transferases/metabolismo , Proteínas de Plantas/metabolismo , Compostos Orgânicos Voláteis/metabolismo
11.
PLoS Biol ; 19(1): e3001022, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465061

RESUMO

Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-ß-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-ß-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-ß-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-ß-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.


Assuntos
Alquil e Aril Transferases/metabolismo , Afrodisíacos/antagonistas & inibidores , Borboletas , Feromônios/metabolismo , Alquil e Aril Transferases/genética , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Borboletas/genética , Borboletas/metabolismo , Evolução Molecular , Feminino , Genes de Insetos , Masculino , Feromônios/farmacologia , Filogenia , Comportamento Sexual Animal/efeitos dos fármacos , Especificidade da Espécie
12.
Nat Commun ; 11(1): 5273, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077723

RESUMO

The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations in hcis-PT cause severe human diseases. Here, we reveal that hcis-PT exhibits a heterotetrameric assembly in solution, consisting of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and inactive Nogo-B receptor (NgBR) heterodimers. Importantly, the 2.3 Å crystal structure reveals that the tetramer assembles via the DHDDS C-termini as a dimer-of-heterodimers. Moreover, the distal C-terminus of NgBR transverses across the interface with DHDDS, directly participating in active-site formation and the functional coupling between the subunits. Finally, we explored the functional consequences of disease mutations clustered around the active-site, and in combination with molecular dynamics simulations, we propose a mechanism for hcis-PT dysfunction in retinitis pigmentosa. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.


Assuntos
Alquil e Aril Transferases/química , Receptores de Superfície Celular/química , Retinite Pigmentosa/genética , Transferases/química , Transferases/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Dimerização , Humanos , Mutação , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Retinite Pigmentosa/enzimologia , Transferases/metabolismo
13.
Chemosphere ; 254: 126911, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32957300

RESUMO

Trivalent organoarsenicals such as methylarsenite (MAs(III)) are considerably more toxic than inorganic arsenate (As(V)) or arsenite (As(III)). In microbial communities MAs(III) exhibits significant antimicrobial activity. Although MAs(III) and other organoarsenicals contribute to the global arsenic biogeocycle, how they exert antibiotic-like properties is largely unknown. To identify possible targets of MAs(III), a genomic library of the gram-negative bacterium, Shewanella putrefaciens 200, was expressed in Escherichia coli with selection for MAs(III) resistance. One clone contained the S. putrefaciens murA gene (SpmurA), which catalyzes the first committed step in peptidoglycan biosynthesis. Overexpression of SpmurA conferred MAs(III) resistance to E. coli. Purified SpMurA was inhibited by MAs(III), phenylarsenite (PhAs(III)) or the phosphonate antibiotic fosfomycin but not by inorganic As(III). Fosfomycin inhibits MurA by binding to a conserved residue that corresponds to Cys117 in SpMurA. A C117D mutant was resistant to fosfomycin but remained sensitive to MAs(III), indicating that the two compounds have different mechanisms of action. New inhibitors of peptidoglycan biosynthesis are highly sought after as antimicrobial drugs, and organoarsenicals represent a new area for the development of novel compounds for combating the threat of antibiotic resistance.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Farmacorresistência Bacteriana/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Peptidoglicano/biossíntese , Shewanella putrefaciens/efeitos dos fármacos , Alquil e Aril Transferases/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Peptidoglicano/metabolismo , Shewanella putrefaciens/genética
14.
Proc Natl Acad Sci U S A ; 117(34): 20794-20802, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32817466

RESUMO

Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme's active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme's active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Transferases/química , Transferases/metabolismo , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Domínio Catalítico , Cromatografia Líquida de Alta Pressão/métodos , Cristalografia por Raios X , Glicosilação , Humanos , Mutação , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores de Superfície Celular/genética , Relação Estrutura-Atividade , Transferases/genética
15.
J Oleo Sci ; 69(7): 711-718, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612020

RESUMO

Angelica keiskei (ashitaba) is an edible plant belonging to the Apiacea family. We focused on sesquiterpenes in the leaves eaten by humans (specifically, in the Japanese population), and confirmed the presence of several sesquiterpenes by GC-MS. Thus, total RNA was extracted from the ashitaba leaves, reverse transcribed, and the resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. Consequently, we were able to isolate two full-length Tps genes (designated AkTps1 and AkTps2). Functional analysis of these two genes was carried out with Escherichia coli cells that expressed mevalonate pathway genes to increase the substrate (farnesyl diphosphate) amount of sesquiterpene synthase, revealing that AkTps1 encodes germacrene D synthase, and AkTps2 codes for an enzyme that catalyzes the generation of germacrene B and smaller amounts of germacrene D (a germacrene B and D synthase). We proposed biosynthetic routes of these two sesquiterpenes from farnesyl diphosphate (FPP) via farnesyl cation.


Assuntos
Angelica/genética , Angelica/metabolismo , Clonagem Molecular/métodos , DNA Circular , Glucosiltransferases/isolamento & purificação , Folhas de Planta/química , Folhas de Planta/genética , RNA de Plantas/isolamento & purificação , Sesquiterpenos/análise , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Catálise , Escherichia coli , Cromatografia Gasosa-Espectrometria de Massas , Amplificação de Genes , Ácido Mevalônico/metabolismo , Reação em Cadeia da Polimerase , Sesquiterpenos de Germacrano/metabolismo , Transdução de Sinais/genética
16.
PLoS One ; 15(7): e0235416, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614884

RESUMO

Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60-70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems.


Assuntos
Alquil e Aril Transferases , Proteínas de Plantas , Plectranthus/enzimologia , Monoterpenos Acíclicos/metabolismo , Alquil e Aril Transferases/biossíntese , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Clonagem Molecular , Escherichia coli/genética , Folhas de Planta/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo
17.
Plant Mol Biol ; 104(1-2): 203-215, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32683610

RESUMO

KEY MESSAGE: Distinct catalytic features of the Poaceae TPS-a subfamily arose early in grass evolution and the reactions catalyzed have become more complex with time. The structural diversity of terpenes found in nature is mainly determined by terpene synthases (TPS). TPS enzymes accept ubiquitous prenyl diphosphates as substrates and convert them into the various terpene skeletons by catalyzing a carbocation-driven reaction. Based on their sequence similarity, terpene synthases from land plants can be divided into different subfamilies, TPS-a to TPS-h. In this study, we aimed to understand the evolution and functional diversification of the TPS-a subfamily in the Poaceae (the grass family), a plant family that contains important crops such as maize, wheat, rice, and sorghum. Sequence comparisons showed that aside from one clade shared with other monocot plants, the Poaceae TPS-a subfamily consists of five well-defined clades I-V, the common ancestor of which probably originated very early in the evolution of the grasses. A survey of the TPS literature and the characterization of representative TPS enzymes from clades I-III revealed clade-specific substrate and product specificities. The enzymes in both clade I and II function as sesquiterpene synthases with clade I enzymes catalyzing initial C10-C1 or C11-C1 ring closures and clade II enzymes catalyzing C6-C1 closures. The enzymes of clade III mainly act as monoterpene synthases, forming cyclic and acyclic monoterpenes. The reconstruction and characterization of clade ancestors demonstrated that the differences among clades I-III were already present in their ancestors. However, the ancestors generally catalyzed simpler reactions with less double-bond isomerization and fewer cyclization steps. Overall, our data indicate an early origin of key enzymatic features of TPS-a enzymes in the Poaceae, and the development of more complex reactions over the course of evolution.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Poaceae/enzimologia , Poaceae/genética , Alquil e Aril Transferases/classificação , Clonagem Molecular , Escherichia coli/genética , Evolução Molecular , Genes de Plantas/genética , Liases Intramoleculares/metabolismo , Proteínas de Plantas/genética , Análise de Sequência , Terpenos/metabolismo
18.
Nat Commun ; 11(1): 3653, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694534

RESUMO

The vasculature represents a highly plastic compartment, capable of switching from a quiescent to an active proliferative state during angiogenesis. Metabolic reprogramming in endothelial cells (ECs) thereby is crucial to cover the increasing cellular energy demand under growth conditions. Here we assess the impact of mitochondrial bioenergetics on neovascularisation, by deleting cox10 gene encoding an assembly factor of cytochrome c oxidase (COX) specifically in mouse ECs, providing a model for vasculature-restricted respiratory deficiency. We show that EC-specific cox10 ablation results in deficient vascular development causing embryonic lethality. In adult mice induction of EC-specific cox10 gene deletion produces no overt phenotype. However, the angiogenic capacity of COX-deficient ECs is severely compromised under energetically demanding conditions, as revealed by significantly delayed wound-healing and impaired tumour growth. We provide genetic evidence for a requirement of mitochondrial respiration in vascular endothelial cells for neoangiogenesis during development, tissue repair and cancer.


Assuntos
Mitocôndrias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/patologia , Neovascularização Fisiológica , Cicatrização/fisiologia , Trifosfato de Adenosina/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Linhagem Celular Tumoral/transplante , Respiração Celular , Modelos Animais de Doenças , Embrião de Mamíferos , Desenvolvimento Embrionário/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Neoplasias/irrigação sanguínea , Fosforilação Oxidativa
19.
PLoS One ; 15(4): e0232220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353014

RESUMO

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


Assuntos
Alquil e Aril Transferases/genética , Bactérias/genética , Vias Biossintéticas/genética , Genoma Bacteriano/genética , Filogenia , Terpenos/metabolismo
20.
J Plant Physiol ; 249: 153166, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32422487

RESUMO

Carotenoids are plastid isoprenoid pigments that play critical roles in light harvesting, photoprotection, and phytohormone biosynthesis. They are also vitamin-A precursors and antioxidant molecules important for human nutrition. Apples (e.g. Malus x domestica Borkh), one of the most widely consumed fruits with high nutrient levels, have a very low carotenoid concentration in flesh, compared with other fruits and vegetables. This could be explained by a deficiency in carotenoid synthesis/accumulation and/or accelerated degradation. We analysed the contribution of M. domestica cv. 'Fuji' phytoene synthase (PSY) in the biosynthesis of carotenoids and determined that among four MdPSY genes present in the organism, MdPSY2 and MdPSY5 are highly expressed in leaves and during fruit ripening in line with an increment in carotenoid content in fruits. Furthermore, two representative polymorphic MdPSY2 variants were found, one with a Tyr358Phe substitution (MdPSY2_F) and the other that additionally has a six-amino-acid deletion in the signal peptide (MdPSY2_CG). MdPSY2, MdPSY5, MdPSY2_F and MdPSY2_CG are all localised in plastids. Interestingly, the polymorphic MdPSY2_F and MdPSY2_CG variants show lower enzymatic activity than the wild-type form in a heterologous complementation assay, which could be attributed to the Tyr358Phe substitution close to the active-site pocket, as was suggested by 3-D modelling analysis. The presence of polymorphic MdPSY2 variants with lower enzymatic activity could be partially responsible for the low carotenoid content in Fuji apple fruits.


Assuntos
Alquil e Aril Transferases/genética , Malus/genética , Proteínas de Plantas/genética , Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Simulação por Computador , Malus/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência
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