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1.
J Agric Food Chem ; 68(5): 1364-1372, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31903751

RESUMO

Arbutin, a glycoside, is derived from the leaves of several plants, including wheat, pear, and bearberry plants, and has a significant role in the treatment of melanoma, cystitis, and cough. Here, we aimed to modify Yarrowia lipolytica to produce arbutin. To construct the arbutin synthetic pathway in Y. lipolytica, three genes (chorismate pyruvate-lyase (UbiC), 4-hydroxybenzoate 1-hydroxylase (MNX1), and hydroquinone glucosyltransferase (AS)) were codon-optimized and heterologously expressed. To maximize arbutin production, seven arbutin-biosynthesis molecular targets were overexpressed, and we found that the individual strengthening of DHS1 and DHS2 led to an 8.9- and 7.8-fold improvement in arbutin yield, respectively. Through optimization, a maximum arbutin titer of 8.6 ± 0.7 g/L was achieved using the finally engineered strain, po1f-At09. Overall, this is the first report of heterologous arbutin synthesis in Y. lipolytica at a high titer. Furthermore, this work opens a possibility for the overproduction of shikimate pathway derivatives in Y. lipolytica.


Assuntos
Arbutina/biossíntese , Yarrowia/genética , Yarrowia/metabolismo , Arbutina/química , Engenharia Metabólica , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo , Yarrowia/química
2.
Int J Mol Sci ; 20(23)2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810184

RESUMO

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi (LkCSE) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of Km and Vmax of CSE for caffeoyl shikimate were 98.11 µM and 14.44 nM min-1, respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.


Assuntos
Proteínas de Arabidopsis/química , Hidrolases de Éster Carboxílico/química , Larix/enzimologia , Lignina/biossíntese , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Cycadopsida/enzimologia , Cycadopsida/genética , Regulação da Expressão Gênica de Plantas , Larix/genética , Lignina/genética , Filogenia , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Ácido Chiquímico/química
3.
Phytochemistry ; 164: 236-242, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31185420

RESUMO

Mangiterpenes A-C and 2',3'-seco-manginoid C, four undescribed sesquiterpene/monoterpene-shikimate-conjugated meroterpenoids with spiro ring systems, were isolated from Guignardia mangiferae. The structures and absolute configurations of these compounds were established by comprehensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. Mangiterpenes A-C represent the first examples of sesquiterpene-shikimate-conjugated spirocyclic meroterpenoids, and 2',3'-seco-manginoid C features an unexpected 2',3'-seco-manginoids skeleton. Mangiterpene C strongly inhibited the production of NO inducted by LPS, with an IC50 value of 5.97 µM. It showed an anti-inflammatory effect by means of blocking in the NF-κB signaling pathway and decreasing the expression of inflammatory mediators.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Monoterpenos/farmacologia , Ácido Chiquímico/farmacologia , Compostos de Espiro/farmacologia , Terpenos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Relação Dose-Resposta a Droga , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Monoterpenos/química , Monoterpenos/isolamento & purificação , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Células RAW 264.7 , Ácido Chiquímico/química , Ácido Chiquímico/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Relação Estrutura-Atividade , Terpenos/química , Terpenos/isolamento & purificação
4.
Toxicol Lett ; 312: 65-71, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31048002

RESUMO

Shikimic acid (SA), a widely-known hydroaromatic compound enriched in Bracken fern and Illicium verum (also known as Chinese star anise), increases the risk of gastric and esophageal carcinoma, nevertheless, the influence of SA on breast cancer remains indistinct. Herein we found that, with models in vitro, SA significantly promoted estrogen receptor(ER) positive cells proliferation and NF-κB activation was involved in it. Moreover, our data showed that IκBα, a critically endogenous inhibitor of NF-κB, was repressed. Subsequently, we found increase of miR-300 by SA treatment sand miR-300 could target IκBα mRNA. Additionally, inhibition of miR-300 abrogated the repression of IκBα by SA. As a result, miR-300 was also involved in NF-κB activation and breast cancer cells proliferation promotion due to SA exposure. Taken together, with ER-positive breast cancer cell models in vitro, MCF-7 and T47D, our results implied that SA promoted breast cancer cells proliferation via a miR-300-induced NF-κB dependent pathway controlling cell cycle proteins.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , NF-kappa B/metabolismo , Receptores Estrogênicos/metabolismo , Ácido Chiquímico/farmacologia , Neoplasias da Mama/tratamento farmacológico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/genética , Receptores Estrogênicos/genética , Ácido Chiquímico/química , Ácido Chiquímico/uso terapêutico , Transdução de Sinais/efeitos dos fármacos
5.
Int J Biol Macromol ; 122: 1212-1216, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30227208

RESUMO

This study investigated the impact of Shikimic Acid (SA) obtained from leaves of Artemisia absinthium on protein glycation in the retina of diabetic rats. The GC/MS analysis of A. absinthium showed that the most abundant bioactive compound was SA (C7H10O5) with a measured retention Index (RI) of 1960 compared to that of the reference sample (1712). Male albino rats were divided into two main groups, Group I (control) and Group II (diabetic); Group II was further divided into four subgroups: Group IIa (diabetic control), Group IIb (diabetic rats were given SA orally [50 mg/kg, body weight (bw)/day], Group IIc diabetic rats were given SA orally [100 mg/kg, bw/day], and Group IId (diabetic rats were given metformin orally [100 mg/kg, bw/day] as positive control). The data obtained suggested that SA reduced glucose and glycated hemoglobin levels. In addition, SA also decreased the formation of glucose-derived advanced glycation end products. Interestingly, SA showed interference with the release of inflammatory mediators in retina and possess antioxidant potential. In conclusion, SA protected the tissues from detrimental effects of hyperglycemia and enhanced antioxidant activity. SA could be a potential lead in the process of drug development in the future to prevent retinopathy in diabetic subjects.


Assuntos
Artemisia absinthium/química , Diabetes Mellitus Experimental/metabolismo , Proteínas/metabolismo , Ácido Chiquímico/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Glicemia/metabolismo , Glicosilação/efeitos dos fármacos , Masculino , Folhas de Planta/química , Ratos , Retina/efeitos dos fármacos , Retina/metabolismo , Ácido Chiquímico/química
6.
Pak J Pharm Sci ; 31(6): 2329-2332, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30473500

RESUMO

Isopropylidene shikimic acid (ISA), a new drug derviatived from Shikimic Acid, had been proved to be effective in the cerebral protection after cerebral ischemia and reperfusion. But there was little research on the physical pharmacy and biopharmaceutical properties about the drug. In order to provide some useful data for the pharmaceutical development of ISA, the solubility, stability and Oil/Water partition coefficient (LogP) were determined by the classic preformulation study method, and the transmembrane performance of ISA was studied by Franz -diffusion cell method in vitro. The results showed that ISA was water-soluble with a solubility 32.52mg/ml, which could be improved to 44.32 mg/ml by 1% (w/v) sodium dodecylsulfate; the LogP was -0.63; ISA was less stable in water but it was stable when pH greater than 6.0 and unstable when pH less than 6.0; the accumulated permeation rates at 1h were about 50% and more than 80% at 6h. Data obtained by the study indicated that the medium selection and pH control were important for liquid preparation of ISA, and avoiding dissolution and absorption in stomach was critical for the oral solid dosage forms. Mucosal drug delivery systems would be considered, according to the certain hydrophilic-lipophilic characters and good transmembrane capability.


Assuntos
Fármacos Neuroprotetores/química , Ácido Chiquímico/química , Composição de Medicamentos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Excipientes/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fármacos Neuroprotetores/farmacologia , Permeabilidade , Ácido Chiquímico/análogos & derivados , Ácido Chiquímico/farmacologia , Dodecilsulfato de Sódio/química , Solubilidade , Solventes/química , Água/química
7.
Biosci Rep ; 38(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30242059

RESUMO

In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/química , Regulação Alostérica/genética , Pseudomonas aeruginosa/enzimologia , Piocianina/biossíntese , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Sequência de Aminoácidos/genética , Sítios de Ligação , Cristalografia por Raios X , Fosfatos/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Piocianina/química , Piocianina/genética , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo
8.
Molecules ; 23(6)2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29895756

RESUMO

We describe the syntheses of nine new angucyclinone 6-aza-analogues, achieved through a hetero Diels-Alder reaction between the shikimic acid derivative-azadiene 13, with different naphthoquinones. The cytotoxic activity of the new synthesized compounds and five angucyclinones, previously reported, was evaluated in vitro against three cancer cell lines: PC-3 (prostate cancer), HT-29 (colon cancer), MCF-7 (breast cancer), and one non-tumoral cell line, human colon epithelial cells (CCD841 CoN). Our results showed that most 6-azadiene derivatives exhibited significant cytotoxic activities, which was demonstrated by their IC50 values (less than 10 µM), especially for the most sensitive cells, PC-3 and HT-29. From a chemical point of view, depending on the protected group of ring A and the pattern of substitution on ring D, cytotoxicity elicited these compounds, in terms of their potency and selectivity. Therefore, according to these chemical features, the most promising agents for every cancer cell line were 7a, 17, and 19c for PC-3 cells; 7a, 17, and 20 for HT-29 cells, and 19a for MCF-7 cells.


Assuntos
Antraquinonas/síntese química , Antineoplásicos/síntese química , Ácido Chiquímico/química , Antraquinonas/química , Antraquinonas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reação de Cicloadição , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Humanos , Células MCF-7 , Estrutura Molecular , Relação Estrutura-Atividade
9.
Chem Biol Drug Des ; 92(2): 1468-1474, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29676519

RESUMO

In this study, we describe the development of new machine learning models to predict inhibition of the enzyme 3-dehydroquinate dehydratase (DHQD). This enzyme is the third step of the shikimate pathway and is responsible for the synthesis of chorismate, which is a natural precursor of aromatic amino acids. The enzymes of shikimate pathway are absent in humans, which make them protein targets for the design of antimicrobial drugs. We focus our study on the crystallographic structures of DHQD in complex with competitive inhibitors, for which experimental inhibition constant data is available. Application of supervised machine learning techniques was able to elaborate a robust DHQD-targeted model to predict binding affinity. Combination of high-resolution crystallographic structures and binding information indicates that the prevalence of intermolecular electrostatic interactions between DHQD and competitive inhibitors is of pivotal importance for the binding affinity against this enzyme. The present findings can be used to speed up virtual screening studies focused on the DHQD structure.


Assuntos
Hidroliases/metabolismo , Aprendizado de Máquina , Área Sob a Curva , Sítios de Ligação , Humanos , Hidroliases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Curva ROC , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo , Eletricidade Estática
10.
Biochemistry ; 57(18): 2667-2678, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29608284

RESUMO

The shikimate pathway is responsible for the biosynthesis of key aromatic metabolites in microorganisms and plants. The enzyme 3-deoxy-d- arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the pathway and DAH7PSs are classified as either type I or type II. The DAH7PSs from Pseudomonas aeruginosa are of particular interest as open reading frames encoding four putative DAH7PS isoenzymes, two classified as type Iα and two classified as type II, have been identified. Here, the structure of a type II DAH7PS enzyme from P. aeruginosa (PAO1) has been determined at 1.54 Å resolution, in complex with its allosteric inhibitor tryptophan. Structural differences in the extra-barrel elements, when compared to other type II DAH7PS enzymes, directly relate to the formation of a distinct quaternary conformation with consequences for allosteric function and the control of flux to branching pathways. In contrast to the well-characterized Mycobacterium tuberculosis type II DAH7PS, which binds multiple allosteric inhibitors, this PaeDAH7PSPA2843 is observed to be modestly allosterically inhibited by a single aromatic amino acid, tryptophan. In addition, structures in complex with tyrosine or with no allosteric ligand bound were determined. These structures provide new insights into the linkages between the active and allosteric sites. With four putative DAH7PS enzymes, P. aeruginosa appears to have evolved control of shikimate pathway flux at the genetic level, rather than control by multiple allosteric effectors to a single type II DAH7PS, as in M. tuberculosis. Type II DAH7PSs, thus, appear to have a more varied evolutionary trajectory than previously indicated.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/química , Evolução Molecular , Pseudomonas aeruginosa/enzimologia , Ácido Chiquímico/metabolismo , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Regulação Alostérica/genética , Sítio Alostérico/genética , Sítios de Ligação , Cristalografia por Raios X , Redes e Vias Metabólicas/genética , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Pseudomonas aeruginosa/genética , Ácido Chiquímico/química , Triptofano/química
11.
FEMS Yeast Res ; 18(2)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29462295

RESUMO

A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.


Assuntos
Redes e Vias Metabólicas , Saccharomyces cerevisiae/metabolismo , Ácido Chiquímico/metabolismo , Ácido Sórbico/análogos & derivados , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ácido Chiquímico/química , Ácido Sórbico/química , Ácido Sórbico/metabolismo
12.
J Enzyme Inhib Med Chem ; 33(1): 397-404, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29363372

RESUMO

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC50 588 and 589 µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Escherichia coli/enzimologia , Ácido Chiquímico/farmacologia , Oxirredutases do Álcool/metabolismo , Relação Dose-Resposta a Droga , Modelos Moleculares , Conformação Molecular , Ácido Chiquímico/síntese química , Ácido Chiquímico/química , Relação Estrutura-Atividade
13.
Org Lett ; 19(21): 5956-5959, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29039958

RESUMO

Manginoids A-G (1-7), seven monoterpene-shikimate-conjugated meroterpenoids with a spiro ring system, were isolated from Guignardia mangiferae. Compounds 1-4 are four isomers with epimeric and double-bond isomeric features possessing a 6-oxaspiro[bicyclo[3.2.1]octane-3,5'-indene] ring, which represent the first examples of spiro meroterpenoids bearing a bridged spirocyclohexanedione moiety. Compounds 5 and 6 possess an unexpected 2,4-dioxatricyclo[3.3.1.03,7]nonane motif, which fuses with a 6-oxabicyclo[3.2.1]octane moiety. Compound 1 exhibits inhibitory activities against 11ß-hydroxysteroid dehydrogenase type 1 with an IC50 value of 0.84 µM.


Assuntos
Ácido Chiquímico/química , Ascomicetos , Isomerismo , Estrutura Molecular , Monoterpenos , Terpenos
14.
Biosens Bioelectron ; 98: 457-465, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28715793

RESUMO

Knowledge of intracellular metabolite levels is important for the understanding of metabolic flux distributions. Whole-cell biosensors of key metabolites are ideal for the monitoring of carbon flow in important metabolic pathways, thus guiding metabolic engineering for microbial improvement. However, lack of biosensors for metabolites of interests has limited their applications. In this study, a genetically encoded whole-cell biosensor specifically responding to shikimic acid has been developed by screening a site-saturation mutagenesis library of the binding pocket of a uric acid-responsive regulatory protein. This biosensor has been successfully applied in analyzing and engineering metabolic flux in the shikimic acid pathway, through genome-wide screening of gene targets critical for the pathway flux, and by improving the specific activity of pathway key enzyme, AroG. This work demonstrates the feasibility of monitoring metabolic flux with the aid of whole-cell biosensors designed for key metabolites.


Assuntos
Técnicas Biossensoriais , Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Ácido Chiquímico/isolamento & purificação , Carbono/química , Escherichia coli , Engenharia Metabólica , Ácido Chiquímico/química , Fatores de Transcrição
15.
Mar Drugs ; 15(1)2017 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-28124983

RESUMO

Pericosine E (6), a metabolite of Periconia byssoides OUPS-N133 was originally isolated from the sea hare Aplysia kurodai, which exists as an enantiomeric mixture in nature. The enantiospecific syntheses of both enantiomers of Periconia byssoides OUPS-N133 has been achieved, along with six stereoisomers, using a common simple synthetic strategy. For these efficient syntheses, highly regio- and steroselective processes for the preparation of bromohydrin and anti-epoxide intermediates were applied. In order to access the unique O-linked carbadisaccharide structure, coupling of chlorohydrin as a donor and anti-epoxide as an acceptor was achieved using catalytic BF3·Et2O. Most of the synthesized compounds exhibited selectively significant inhibitory activity against α-glycosidase derived from yeast. The strongest analog showed almost 50 times the activity of the positive control, deoxynojirimycin.


Assuntos
Dissacarídeos/química , Inibidores de Glicosídeo Hidrolases/química , Ácido Chiquímico/análogos & derivados , alfa-Glucosidases/química , 1-Desoxinojirimicina/química , Álcoois/química , Ascomicetos/química , Cloridrinas/química , Compostos de Epóxi/química , Glucosamina/análogos & derivados , Glucosamina/química , Ácido Chiquímico/química , Estereoisomerismo , Leveduras/química
16.
Biochemistry ; 56(4): 592-601, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28045507

RESUMO

3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with Ki = 1.5 µM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KM,MnKM,PEPKM,E4P/kcat) versus log(Ki), revealed a slope, m, of 0.34, with r2 = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Oximas/química , Proteínas Recombinantes de Fusão/química , Fosfatos Açúcares/química , 3-Desoxi-7-Fosfo-Heptulonato Sintase/química , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Mimetismo Molecular , Mutação , Fosfoenolpiruvato/química , Fosfoenolpiruvato/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo , Fosfatos Açúcares/metabolismo , Termodinâmica
17.
Mater Sci Eng C Mater Biol Appl ; 71: 167-175, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27987694

RESUMO

Shikimic acid (SA) is a key raw material for the synthesis of the antiviral drug, but its extraction and separation from plants is still limited. Crosslinked poly (acryloyloxyethyltrimethyl ammonium chloride, DAC) microspheres were synthesized via inverse-phase suspension polymerization. In the synthesizing, N,N'-methylene bisacrylamide (MBA) was used as crosslinker, cyclohexane as dispersed medium and span-60 as dispersants, obtaining CPDAC gel microspheres. The effect of polymerization condition on balling performance and the characteristics of CPDAC were examined. The adsorption properties of CPDAC towards SA were mainly explored and the data of adsorption isotherm were analyzed by using Langmuir, Freundlich, Temkin, Sips and Toth models. Furthermore, the adsorption mechanism was analyzed in depth, and the adsorption thermodynamics was also investigated. The results show that in order to prepare CPDAC, water phase must be added dropwise to oil phase, and the volume ratio of oil-water is more than 2:1. The mean diameter of CPDAC decreases with increasing span-60 and accelerating agitating rate. The strong electrostatic interaction is formed between quaternary ammonium nitrogen of CPDAC and COO- of SA. The adsorption kinetic data is fitted well with pseudo-first-order model. The adsorption ability is higher in aqueous water than ethanol, reaching 108mg/g, and Toth model is more suitable for describing the actual adsorption process. The adsorption of CPDAC towards SA is dependent on the pH value of the medium. The adsorption process is exothermic, the adsorption amount decreases with the increase of temperature, and the process is driven by enthalpy. The adsorption amount decreases with the increase of salinity. The reusability of CPDAC towards SA can keep 86.1% at the sixth cycle.


Assuntos
Microesferas , Modelos Químicos , Ácido Chiquímico/química , Adsorção
18.
Mini Rev Med Chem ; 17(12): 1013-1027, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27342231

RESUMO

OBJECTIVE: There has been a massive increase in the number of reports about the medicinal benefits of the consumption of phenylpropanoids derived from the plastidic shikimate pathway. These benefits include anti-retroviral, anti-hypertensive, anti-inflammatory, anti-aging and insulin-sensitizing activities, the reduction of the risk of a range of chronic diseases including cardiovascular disease, cancer and osteoporosis as well as inhibition of LDL (low-density lipoprotein) oxidation. In addition, chorismate-derived salicylate which was originally isolated from plants, albeit now under chemical production, is massively used for pain relief in the form of acetylsalycilic acid, namely aspirin. Chorismate also acts as precursor in the biosynthesis of folate and phylloquinone, i.e., vitamins B9 and K1, respectively. RESULTS: Cumulative evidence suggests that deficiencies of either of these vitamins in the diet can result in a wide range of diseases. In parallel to our enhanced comprehension of the dietary importance of shikimate-derived compounds, the advent of metabolomics and the development of next-generation sequencing technologies have dramatically accelerated advances in our understanding of the biosynthetic, decorative and degradation pathways underlying their metabolism. Furthermore, forward and reverse genetic approaches have begun to facilitate the metabolic engineering of plants for biofortification of these compounds. CONCLUSION: Here we review data about the bioactivities of these compounds and provide an overview of our current understanding of biosynthesis, molecular function and their in planta occurrence. Finally we discuss the future perspectives and the importance of further development of cross-disciplinary research efforts in this rapidly expanding research field.


Assuntos
Flavonoides/biossíntese , Propanóis/metabolismo , Ácido Chiquímico/metabolismo , Ácido Corísmico/biossíntese , Ácido Corísmico/química , Flavonoides/química , Engenharia Metabólica , Plantas/química , Plantas/metabolismo , Propanóis/química , Salicilatos/química , Salicilatos/metabolismo , Ácido Chiquímico/química
19.
J Agric Food Chem ; 64(51): 9663-9674, 2016 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-27981846

RESUMO

Coffee is one of the most consumed beverages in the world, due to its unique aroma and stimulant properties. Although its health effects are controversial, moderate intake seems to be beneficial. The present work deals with the characterization and quantification of polyphenols and methylxanthines in four Arabica green coffee beans from different geographical origins. The antioxidant activity was also evaluated. Forty-three polyphenols (cinnamic acid, cinnamoyl-amide, 5 cinammoyl-glycosides, and 36 cinnamate esters) were identified using LC-MSn. Among these, cinnamate esters of six different chemical groups (including two dimethoxycinnamoylquinic acid isomers, three caffeoyl-feruloylquinic acid isomers, caffeoyl-sinapoylquinic acid, p-coumaroyl-feruloylquinic acid, two caffeoylshikimic acid isomers, and trimethoxycinnamoylshikimic acid) in addition to five isomers of cinnamoyl-glycosides called caffeoyl-2,7-anhydro-3-deoxy-2-octulopyranosic acid (CDOA) are described for the first time in Arabica green coffee beans. Moreover, 38 polyphenols (6-7% w/w) and 2 methylxanthines (1.3% w/w) were quantified by HPLC-DAD. Caffeoylquinic was the most abundant group of compounds (up to 85.5%) followed by dicaffeoylquinic and feruloylquinic acids (up to 8 and 7%, respectively) and the newly identified cinnamoyl-glycosides (CDOA) (up to 2.5%). Caffeine was the main methylxanthine (99.8%), with minimal amounts of theobromine (0.2%). African coffees (from Kenya and Ethiopia) showed higher polyphenolic content than American beans (from Brazil and Colombia), whereas methylxanthine contents varied randomly. Both phenols and methylxanthines contributed to the antioxidant capacity associated with green coffee, with a higher contribution of polyphenols. We conclude that green coffee represents an important source of polyphenols and methylxanthines, with high antioxidant capacity.


Assuntos
Coffea/química , Extratos Vegetais/química , Polifenóis/química , Glicosídeos/química , Espectrometria de Massas , Estrutura Molecular , Extratos Vegetais/isolamento & purificação , Polifenóis/isolamento & purificação , Sementes/química , Ácido Chiquímico/química
20.
Biochemistry ; 55(45): 6314-6326, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27805809

RESUMO

Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is an essential acyltransferase that mediates flux through plant phenylpropanoid metabolism by catalyzing a reaction between p-coumaroyl-CoA and shikimate, yet it also exhibits broad substrate permissiveness in vitro. How do enzymes like HCT avoid functional derailment by cellular metabolites that qualify as non-native substrates? Here, we combine X-ray crystallography and molecular dynamics to reveal distinct dynamic modes of HCT under native and non-native catalysis. We find that essential electrostatic and hydrogen-bonding interactions between the ligand and active site residues, permitted by active site plasticity, are elicited more effectively by shikimate than by other non-native substrates. This work provides a structural basis for how dynamic conformational states of HCT favor native over non-native catalysis by reducing the number of futile encounters between the enzyme and shikimate.


Assuntos
Aciltransferases/química , Proteínas de Arabidopsis/química , Domínio Catalítico , Conformação Proteica , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação/genética , Biocatálise , Cristalografia por Raios X , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Mutação , Homologia de Sequência de Aminoácidos , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo , Eletricidade Estática , Especificidade por Substrato
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