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1.
Enzyme Microb Technol ; 120: 69-76, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30396401

RESUMO

Nordihydroguaiaretic acid (NDGA) is the major lignan of the creosote bush Larrea tridentata known for its antioxidative and pharmacological properties. Here we present the identification of glucansucrases for NDGA glucosylation and the physicochemical and biological characterization of the glucosides. Extracellular glucansucrase of L. pseudomesenteroides DSM 20193 was selected from 19 glucansucrase positive Leuconostoc and Weissella strains. Kinetic analysis of the PEG-fractionated enzyme revealed a KM of 6.6 mM and a kcat of 2.6 s-1 for NDGA. Full-factorial design methodology was used to optimize conversion resulting in 95.5% total NDGA glucosides. In total 7 glucosides were detected by LC-MS ranging from mono- to triglucoside. The 4-O-α-D-monoglucoside and the symmetrical 4,4'-O-α-D-diglucoside were the major products in all biotransformations. Water solubility and half-life stability at 45 °C increased significantly in the order diglucoside > monoglucoside > aglycon. Analysis of cellular antioxidative capacity exhibited a time-dependent activity increase pointing towards glucoside hydrolysis. Accordingly, NDGA-glucosides impaired metastasis of triple negative breast cancer cells to the same degree as the aglycon with 35% reduction of cell migration by the mono- and 34% reduction by the diglucoside after 20 h.


Assuntos
Antioxidantes/farmacologia , Glucosídeos/síntese química , Glucosídeos/farmacologia , Glicosiltransferases/metabolismo , Larrea/enzimologia , Masoprocol/química , Neoplasias de Mama Triplo Negativas/patologia , Antioxidantes/síntese química , Movimento Celular , Feminino , Humanos , Glucosídeos Iridoides , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Células Tumorais Cultivadas
2.
Arch Biochem Biophys ; 541: 37-46, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24189289

RESUMO

The creosote bush (Larrea tridentata) harbors members of the monolignol acyltransferase, allylphenol synthase, and propenylphenol synthase gene families, whose products together are able to catalyze distinct regiospecific conversions of various monolignols into their corresponding allyl- and propenyl-phenols, respectively. In this study, co-expression of a monolignol acyltransferase with either substrate versatile allylphenol or propenylphenol synthases in Escherichia coli established that various monolignol substrates were efficiently converted into their corresponding allyl/propenyl phenols, as well as providing proof of concept for efficacious conversion in a bacterial platform. This capability thus potentially provides an alternate source to these important plant phytochemicals, whether for flavor/fragrance and fine chemicals, or ultimately as commodities, e.g., for renewable energy or other intermediate chemical purposes. Previous reports had indicated that specific and highly conserved amino acid residues 84 (Phe or Val) and 87 (Ile or Tyr) of two highly homologous allyl/propenyl phenol synthases (circa 96% identity) from a Clarkia species mainly dictate their distinct regiospecific catalyzed conversions to afford either allyl- or propenyl-phenols, respectively. However, several other allyl/propenyl phenol synthase homologs isolated by us have established that the two corresponding amino acid 84 and 87 residues are not, in fact, conserved.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Eugenol/análogos & derivados , Larrea/enzimologia , Oxirredutases/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Eugenol/química , Eugenol/metabolismo , Engenharia Genética , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/genética , Estereoisomerismo , Especificidade por Substrato
3.
Immunopharmacol Immunotoxicol ; 34(2): 346-53, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22268619

RESUMO

Larrea divaricata Cav. (Jarilla) is a bush widely used in folk therapy for the treatment of several pathologies. Partially purified proteins of crude extract (JPCE) cross-react with proteins of Gram-negative bacteria, including Pseudomonas aeruginosa, which is an opportunistic pathogen that causes several intrahospitalary infections. This bacterium produces many proteins with enzymatic activity, including hemolysins and proteases that play a major role in acute infection caused by this bacterium. The aim of our work was to investigate if antibodies against with L. divaricata neutralize the hemolytic and proteolytic activity of P. aeruginosa. The hemolytic activity of soluble cellular proteins was inhibited 100% and extracellular proteins (EP) showed an inhibition between 44 and 95% when both bacterial fractions were treated with anti-JPCE serum. Also, in EP the neutralization was directed towards the active site of the hemolysin. When protease activity of extracellular products was tested, bands of 217, 155, 121, 47 and 27 kDa were observed in native zymograms. Neutralization between 55 and 70% of the bands of 217, 155 and 121 kDa was observed when EP were treated with anti-JPCE serum. In conclusion, our data clearly demonstrate that antibodies elicited with L. divaricata' proteins are able to neutralize the hemolytic and proteolytic activity of P. aeruginosa cellular and extracellular proteins. Our study constitutes the first report that associates the immunogenicity of plant proteins and bacterial proteins with enzymatic activity. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.


Assuntos
Anticorpos Neutralizantes/farmacologia , Biocatálise/efeitos dos fármacos , Reações Cruzadas/imunologia , Enzimas/imunologia , Larrea/enzimologia , Proteínas de Plantas/imunologia , Pseudomonas aeruginosa/enzimologia , Animais , Anticorpos Neutralizantes/imunologia , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Antígenos de Plantas/metabolismo , Antígenos de Plantas/farmacologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Extratos Celulares/química , Meios de Cultivo Condicionados/química , Enzimas/metabolismo , Feminino , Hemólise/efeitos dos fármacos , Humanos , Soros Imunes/imunologia , Soros Imunes/farmacologia , Larrea/química , Masculino , Mercaptoetanol/farmacologia , Camundongos , Camundongos Endogâmicos , Peptídeo Hidrolases/imunologia , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Folhas de Planta/enzimologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Caules de Planta/química , Caules de Planta/enzimologia , Inibidores de Proteases/imunologia , Inibidores de Proteases/farmacologia , Desnaturação Proteica/efeitos dos fármacos , Pseudomonas aeruginosa/química , Vacinação/métodos
4.
J Biol Chem ; 286(41): 35683-35688, 2011 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-21880724

RESUMO

The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO(2) fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.


Assuntos
Larrea/enzimologia , Modelos Moleculares , Proteínas de Plantas/química , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
5.
Arch Biochem Biophys ; 465(1): 209-18, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17624297

RESUMO

The creosote bush (Larrea tridentata) accumulates a complex mixture of 8-8' regiospecifically linked lignans, of which the potent antioxidant nordihydroguaiaretic acid (NDGA) is the most abundant. Its tetra-O-methyl derivative (M4N) is showing considerable promise in the treatment of refractory (hard-to-treat) brain and central nervous system tumors. NDGA and related 9,9'-deoxygenated lignans are thought to be formed by dimerization of allyl/propenyl phenols, phenylpropanoid compounds that lack C-9 oxygenation, thus differentiating them from the more common monolignol-derived lignans. In our ongoing studies dedicated towards elucidating the biochemical pathway to NDGA and its congeners, a pinoresinol-lariciresinol reductase homologue was isolated from L. tridentata, with the protein obtained in functional recombinant form. This protein efficiently catalyzes the conversion of p-coumaryl and coniferyl alcohol esters into the corresponding allylphenols, chavicol and eugenol; neither of their propenylphenol regioisomers, p-anol and isoeugenol, are formed during this enzyme reaction.


Assuntos
Anisóis/química , Eugenol/química , Isoenzimas/química , Larrea/enzimologia , Oxirredutases/química , Fenóis/química , Propionatos/química , Derivados de Alilbenzenos , Sequência de Aminoácidos , Catálise , Ácidos Cumáricos , Ativação Enzimática , Ésteres , Eugenol/análogos & derivados , Isoenzimas/metabolismo , Dados de Sequência Molecular
6.
Proc Natl Acad Sci U S A ; 100(19): 10641-6, 2003 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-12960376

RESUMO

An enantio-specific polyphenol oxidase (PPO) was purified approximately 1,700-fold to apparent homogeneity from the creosote bush (Larrea tridentata), and its encoding gene was cloned. The posttranslationally processed PPO ( approximately 43 kDa) has a central role in the biosynthesis of the creosote bush 8-8' linked lignans, whose representatives, such as nordihydroguaiaretic acid and its congeners, have potent antiviral, anticancer, and antioxidant properties. The PPO primarily engenders the enantio-specific conversion of (+)-larreatricin into (+)-3'-hydroxylarreatricin, with the regiochemistry of catalysis being unambiguously established by different NMR spectroscopic analyses; the corresponding (-)-enantiomer did not serve as a substrate. This enantio-specificity for a PPO, a representative of a widespread class of enzymes, provides additional insight into their actual physiological roles that hitherto have been difficult to determine.


Assuntos
Catecol Oxidase/metabolismo , Larrea/enzimologia , Sequência de Aminoácidos , Catecol Oxidase/química , Catecol Oxidase/genética , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Estereoisomerismo
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